Biophysics Seminar

semester, 2018


Thursday, January 18th 2018
10:10 am:
Biophysics Seminar in PAN 120
Speaker: John Yin, University of Wisconsin
Subject: Paths to biological polymers: an insight from virus infections and origins of life

(1) Given the genome of a virus and PubMed, how well could one predict the one-step growth of the virus? Decades of biochemical and biophysical studies on bacteriophage T7, incorporated into a chemical kinetic model for template-dependent processes of transcription, translation, and genome replication, as well as particle assembly and release, enabled simulation of one-step growth behavior that recapitulated the experimentally observed kinetics of phage growth. Extension of the model and experiments to study the effects of host-cell physiology on phage growth highlighted the host cellular protein synthesis machinery as a key limiting resource for phage growth.
(2) Given amino acid monomers, but no cells, no templates and no protein synthesis machinery, how might the monomers nevertheless form polymers? The synthesis of peptide bonds between amino acids is a condensation reaction that is generally disfavored in aqueous solutions. However, we have found that for appropriate initial conditions of pH and temperature, drying of amino acids can promote their condensation to form peptides.
So what is the common insight from (1) and (2)? The often neglected “nurture” part of “nature versus nurture” can be important. The kinetics of phage growth depends on the physiological state of its host cell, and the de novo synthesis of a polypeptide species critically depends on the acidity and temperature of its initial solution. In short, we are all products of our environments.

Faculty Host: J. Woods Halley

Thursday, January 25th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Ryan Marshall, UMN
Subject: Rapid and scalable characterization of CRISPR technologies using a cell-free transcription-translation system (TXTL)

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. We present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression—all without protein purification or live cells. We use TXTL to measure the dynamics of DNA cleavage and gene repression for single and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of diverse anti-CRISPR proteins, develop a fast and scalable screen for protospacer-adjacent motifs, and show that dCas9 bound to a protospacer can be displaced. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.


Thursday, February 1st 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Joseph Muretta, University of Minnesota, Biochemistry, Molecular Biology and Biophysics
Subject: Adventures in Biophysics: Lysine Acetylation Tunes Mechanochemical Coupling and Force Output in a Mitotic Kinesin

Thursday, February 8th 2018
10:10 am:
Biophysics Seminar in 120 PAN
No Seminar This Week

Thursday, February 15th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jasmine Foo, School of Mathematics, University of Minnesota
Subject: Impact of the tumor microenvironment on drug resistance in cancer

Despite the effectiveness of many therapies in reducing tumor burden during the initial phase of treatment, the emergence of drug resistance remains a primary obstacle in cancer treatment. Tumors are comprised of highly heterogeneous, rapidly evolving cell populations whose dynamics can be modeled using evolutionary theory. In this talk I will describe some mathematical models of the evolutionary processes driving drug resistance in cancer, and demonstrate how these models can be used to provide clinical insights. These models will be applied to study the impact of dosing schedules and the tumor microenvironment on the emergence of drug resistance in lung cancer.


Thursday, February 22nd 2018
10:10 am:
Biophysics Seminar in 120 PAN
No Seminar This Week - Biophysical Society Meeting

Thursday, March 1st 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Tanner Akkin, Associate Professor of Biomedical Engineering, University of Minnesota
Subject: Development of a Serial Optical Coherence Scanner for Visualizing and Mapping the Brain with Microscopic Resolution

The feasibility of mapping and imaging the brain with microscopic resolution is presented. A serial optical coherence scanner, which combines a polarization-sensitive optical coherence tomography and a tissue slicer, distinguishes white matter and gray matter and visualizes nerve fiber tracts that are as small as a few tens of micrometers. The technique utilizes the retardance contrast that arise due to the myelination of nerve fibers and the axis orientation contrast that determine the 2D orientation of the nerve fibers, and the technique can be adapted to measure the inclination angle of the fiber, completing the 3D orientation. This scanner could reveal biomarkers for disease onset and progression, and support development of therapeutics.


Thursday, March 8th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Elizabeth Smith (Elias Puchner lab), School of Physics and Astronomy, University of Minnesota
Subject: Characterization of Ire1 interactions and dynamics with quantitative super-resolution microscopy

Quantitative Super-Resolution Microscopy is a powerful technique to study biological processes below the diffraction limit. In this work, we employ our intracellular calibrated Photoactivated Localization Microscopy (PALM) technique to perform quantitative molecular counting of proteins involved in the unfolded protein response (UPR). The UPR is a signaling pathway which dynamically regulates endoplasmic reticulum (ER) protein folding capacity in response to cellular stress. As is true with many signaling pathways, the spatiotemporal organization of the UPR-specific biomolecules is an inherent feature of the pathway activation and downstream response. Specifically, in response to stress, Ire1 (a bifunctional transmembrane kinase/endoribonuclease) oligomerizes and forms discrete signaling clusters which recruit and splice an mRNA encoding a transcription activator. Using PALM in conjunction with traditional fluorescence microscopy we characterize the interactions and dynamics of Ire1 at wild type expression levels in yeast cells. Specifically, we quantify the oligomeric state, of Ire1 under stressed and unstressed conditions, track the motion of Ire1 during signaling activity, and determine the sensitivity and resolution of spatial cross-correlation in a model system combining traditional and super-resolution fluorescencemicroscoy in the same protein construct (Ire1_yeGFP_mEos2). Finally we perform colocalization experiments with downstream UPR biomolecules to further characterize the role of Ire1 signaling centers in control of gene expression. This study provides insight into the spatiotemporal organization of Ire1 and its downstream partners in the signaling response of the UPR.


Thursday, March 15th 2018
10:10 am:
Biophysics Seminar in 120 PAN
No Seminar This Week (Spring Break)

Thursday, March 22nd 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Yahor Savich (David Thomas lab), School of Physics and Astronomy, University of Minnesota
Subject: Myosin Orientation in a Functioning Muscle Fiber With High Angular Resolution

We have measured the orientation of myosin in a muscle fiber bundle using electron paramagnetic resonance (EPR) and a bifunctional spin label (BSL), with angular resolution of a few degrees. Despite advances in cryo-EM, fluorescence, and small-angle X-ray diffraction, these techniques do not provide high-resolution structural information about myosin heads in vitro under functional conditions. A pair of (i,i+4) Cys residues were engineered on an alpha-helix in the regulatory light chain (RLC). By exchanging endogenous RLC with BSL-labeled RLC on oriented muscle fibers, we were able to resolve angular distributions in several biochemical states due to the stereospecific attachment of BSL’s two disulfide bonds. In this setup, the accurate determination of BSL’s angular coordinates allowed us to determine the orientation of individual structural elements with respect to the muscle fiber axis. Addition of ATP in the absence of Ca, relaxing the muscle, shifted the orientational distribution to a much more disordered distribution. This work is inspired by growing therapeutic interest in super-relaxed myosin state, which predicts presence of order in relaxation.


Thursday, March 29th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Wesley Errington, Molecular Cell Engineering Laboratory, University of Minnesota
Subject: Integrating experimental and computational approaches to elucidate mechanisms of binding in multivalent proteins

Multivalent proteins are ubiquitous in nature and can provide unique, exploitable properties in therapeutic applications such as increased affinity or multi-target specificity. Despite the importance of these proteins in fundamental and applied biomedical research, mechanistic quantitative descriptions of their binding kinetics are limited. We have considered such multivalent protein-protein interactions to be driven by three key variables: the binding affinity of individual monomer units, the linker length/structure between the monomers, and the overall valency of each multivalent protein. Using model synthetic proteins in which all three of these variables can be independently tuned, we have performed surface plasmon resonance experiments to quantify the kinetics of association and dissociation as a function of affinity, linker, and valency. In parallel, we developed a mechanistic model based on mass-action kinetics that explictly enumerates all possible microstates that participate in the binding reaction. Integration of these quantitative experimental and computational approaches has elucidated a number of interesting findings, including the role of valency in generating non-canonical reaction kinetics, that will be discussed. Our approach should enable better understanding of dynamic behaviors in natural multivalent proteins and lead to more rational optimization of multivalent therapeutics.


Thursday, April 5th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: James M. Carothers, Department of Bioengineering, University of Washington
Subject:  Multi-state design of kinetically-controlled RNA aptamer ribosensors

Metabolite-responsive RNA regulators that react to changing conditions through molecular interactions are widespread in biology. In many of these systems, kinetic control mechanisms coordinate co-transcriptional RNA folding with metabolite binding and enable outputs that are highly-sensitive and highly-selective to target ligands. Although synthetic riboswitches exhibiting kinetic control have been identified by chance, it has not been possible to intentionally engineer kinetically-controlled RNA aptamer devices. Consequently, kinetic control mechanisms that could otherwise be exploited to overcome functional limits imposed by the thermodynamics of molecular recognition have remained beyond reach. We recently developed a novel approach for multi-state, co-transcriptional RNA folding design that has allowed us to engineer kinetically-controlled RNA aptamer ribosensors. In this architecture, in vitro selected RNA aptamers are coupled through a timer domain to a toehold-mediated strand displacement (TMSD) actuator such that co-transcriptional ligand-binding generates fluorescence from DNA gates through TMSD. We have shown that ribosensors can be transcribed in situ and used to analyze metabolic production directly from engineered microbial cultures, establishing a new class of cell-free biosensors. We found that kinetically-controlled ribosensors exhibited 5-10 fold greater ligand sensitivity than a thermodynamically-controlled device. And, we further demonstrated that a second aptamer, promiscuous for aromatic amino acid binding, could be assembled into kinetic ribosensors with 45-fold improvements in ligand selectivity. I will present these results and discuss the broader implications of this work for engineering RNA aptamer devices and overcoming thermodynamic constraints on molecular recognition through the design of kinetically-controlled responses.

Faculty Host: Vincent Noireaux

Thursday, April 12th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Aaron Engelhart, Department of Genetics, Cell Biology, and Development, University of Minnesota
Subject:  Hold the water! Probing nucleic acid hydration with alternative solvents

Nucleic acids adopt a myriad of roles in life ranging from their well-known role in simple information transfer (in DNA and mRNA) to functional behaviors including catalysis (in ribozymes including the ribosome) and ligand binding (in riboswitches). The folding of these polymers is intimately connected with the environment afforded by the solvent in which they fold: water. Water dictates the folding behavior of nucleic acids in a variety of ways, including hydrophilic interactions (with the phosphate backbone as well as dissolved cations); nonclassical hydrophobic interactions (which promote base stacking); and minor groove binding of water (as well as cations), which has been known since the first atomic-resolution crystal structures of DNA shown by Drew and Dickerson. We recently showed that, remarkably, nucleic acids can form stable secondary structures in an essentially anhydrous solvent - a so-called "deep eutectic solvent" (DES) formed from a 2:1 molar mixture of urea and choline chloride. Despite the absence of water, a range of secondary structures fold in DES. I will discuss our results using alternative solvents to examine the role of hydration in nucleic acid folding.


Thursday, April 19th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Ashim Rai, (postdoc in Shiv’s lab) Department of Genetics, Cell Biology and Development, University of Minnesota
Subject: Biophysical regulation of myosin VI motility by cargo adaptor proteins

: Cargo transport by motor proteins organizes the cell interior. On cellular cargoes, the binding of motor proteins is mediated by cargo adaptor proteins. Initially thought of as passive scaffolds for motor proteins, cargo adaptor proteins have recently been shown to directly affect motor function through structural studies. However, a biophysical mechanism of cargo adaptor-mediated regulation of motor activity is still lacking. In this study, we have tried to address this problem in the context of the minus-end directed actin motor, Myosin VI. Through direct measurements of adapter-mediated changes in myosin VI motility, conformation and dimerization, we have tried to establish a structure-function relationship between myosin VI and its cargo adaptor proteins. We find that binding to cargo adaptor has a potentiating effect on myosin VI velocity and processivity which is mediated through a combination of auto inhibition release, lever arm extension and dimerization of the myosin VI motor.


Thursday, April 26th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Gianluigi Veglia, Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota
Subject: Role of conformational dynamics on protein kinases function and dysfunction

Eukaryotic protein kinases (EPKs) are phosphoryl transferase that mediate several signaling events and constitute major pharmaceutical targets. cAMP-dependent protein kinase A is a prototypical kinase of paramount biological importance as it is involved in a myriad of cellular processes. Using nuclear magnetic resonance (NMR) spectroscopy, we probed the enzyme’s intramolecular allosteric network along the catalytic cycle. We discovered that kinase A conformational motions are highly organized and correlated during turnover. Fast dynamics in the ps-ns time scale are directly linked to the conformational entropy of binding, revealing the mechanisms for positive and negative allosteric cooperativity that drive both substrate binding and product release. Slow dynamics in the in the micro second to milli second time scale are responsible for the conformational transitions from catalytically incompetent to competent states. Disruption of these dynamics leads to dysfunctional signaling and disease. Since the C-subunit of protein kinase A is highly conserved within the kinase family, the present study offers unprecedented mechanistic insights into intramolecular signaling for designing novel kinase activators or inhibitors.


Thursday, May 3rd 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Ibrahim Cisse, Department of Physics, Massachusetts Institute of Technology
Subject: Super-resolution imaging of transcription in living mammalian cells

Protein clustering is a hallmark of genome regulation in mammalian cells. However, these clusters often emerge from weak or transient interactions between component biomolecules at length scales and with temporal dynamics not readily attained in live cell imaging. My lab focuses on pushing single molecule and super-resolution techniques to enable the detection and characterization of weak or transient biomolecular assemblies with very high spatial and temporal resolutions directly in living mammalian cells. We discovered that very transient clusters of RNA Polymerase II (Pol II) correlate with mRNA synthesis at gene loci. For endogenous β-actin genes in live mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.


Thursday, May 10th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker:  Hyun Youk, Kavli Institute of Nanoscience, TU Delft, Netherlands
Subject: Restarting life on demand: Distinguishing dormancy from death by resuming life in yeast

Stopping life indefinitely and then resuming it at the press of a button is an idea that has captivated movie makers, science fiction writers, and the general public. An open question is whether one can indeed completely stop and then resume, after many years, the life of any animals, including humans. Yeast spores are ideal for investigating the halting and resuming of cellular life. Yeast spores do not outwardly appear to be living – they neither move nor divide, exist without any external energy sources, and are believed to maintain faint, if any, intracellular dynamics. While we know how yeasts form spores when they are starved of nutrients and how a newly introduced energy source (glucose) “wakes-up” the spores and cause them to re-enter replicative life, little is known about the processes that occur, if any, inside the spores during dormancy before glucose is added, how long the dormancy can last (and what determines this timeline), and why some spores cannot wake up (thus considered dead) after a long enough time without any nutrients. We explored these questions by investigating how glucose, the necessary energy source, germinates yeast spores (i.e., restart cell replication). In doing so, we discovered that spores that are conventionally thought to be dead are, in fact, merely dormant. We found that not all spores germinate despite encountering abundant glucose. These un-germinated spores are primed so that they germinate faster upon encountering more glucose. Surprisingly, inducing expression of a useless gene that neither aids nor interferes cell growth, in dormant spores promotes germination. Crucially, we quantify an intrinsic ability by dormant spores to express genes – an intrinsic gene-expression rate. By tuning this, we could tune the probability that a spore germinates and even allow spores that glucose could not germinate, to germinate. Finally, we show that causing spores to use their stored resources to make useless proteins, while dormant, dramatically lengthens by months the time that spores escape death by months. We explain these observations with a simple mathematical model. These results provide quantitative insights into differences between death and dormancy.
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Faculty Host: Elias Puchner

Thursday, October 25th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Oleg Krichevsky, Molecular Biophysics Lab, Ben-Gurion University
Subject:  T cell communication through cytokines follows a simple sink-diffusion model

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. We used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. Using T cell exchange of IL-2 as a model system, we established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80–120 um, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Despite the complexity of the immune organs, the profiles of cytokine fields both in vitro and in vivo quantitatively follow the predictions of a simple model, essentially without any free parameters.

Ref. Oyler-Yaniv A, Oyler-Yaniv J, Whitlock B.M, Liu Z, Germain R.N, Huse M, Altan-Bonnet G. and O. Krichevsky (2017) , Immunity, 46, 609-620.

Faculty Host: Elias Puchner

Thursday, November 15th 2018
10:10 am:
Biophysics Seminar in PAN 120
Speaker: John Marko, Northwestern University
Subject: Single-molecule studies of protein-DNA interactions

All processing of DNA - transcription, replication, recombination and
repair - depend on the interactions of proteins with DNA. I will discuss
single-molecule methods for analyzing protein-DNA interactions, starting
with the (statistical)-mechanical response of DNA molecules and how
monitoring that can allow novel quantitative studies of proteins that fold
and change topology of DNA molecules. I will then describe a phenomenon
that appears pervasive for biomolecule interactions - "facilitated
dissociation" - that makes rates of turnover of molecular complexes in vivo
very different from what we observe in vitro.

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