Biophysics Seminar

semester, 2019


Thursday, January 24th 2019
10:10 am:
Biophysics Seminar in 120 PAN
There will be no seminar this week.

Thursday, January 31st 2019
10:10 am:
Biophysics Seminar in 120 PAN
There will be no seminar this week.

Thursday, February 7th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker:  Karunya Kandimalla, Associate Professor at Department of Pharmaceutics of the University of Minnesota
Subject: Nanovehicles for the Diagnosis and Treatment of Blood-Brain Barrier Dysfunction in Neurodegenerative Diseases

The blood brain barrier (BBB) performs dual functions of restricting the entry of xenobiotics into brain while serving as the major signaling and material trafficking portal between plasma and brain. Moreover, the BBB modulates cerebral blood flow to sustain neuronal activity; handles glucose as well as insulin delivery to brain; and maintains immune and inflammatory communication between periphery and brain. These critical BBB functions are disrupted in neurodegenerative disease like Alzheimer’s disease and cerebral amyloid angiopathy. We developed therapeutic nanoparticles to detect and treat BBB dysfunction in these diseases.
For successful brain delivery, a nanoparticle must withstand dominant clearance pressure from the peripheral reticuloendothelial system, marginate from the bulk blood flow to the vascular endothelium, permeate the blood-brain-barrier, and accumulate at the target site. In addition to these common challenges, the nanoparticles intended for cerebrovascular targeting must incorporate appropriate design elements to ensure their retention in the cerebral vasculature. Furthermore, a functionally optimized nanoparticle design demands synergistic amalgamation of the physicochemical properties of various components and their intended physiological effects. Strategies developed in our laboratory to surmount these barriers will be discussed in the talk.


Thursday, February 14th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jiali Gao, Department of Chemistry, University of Minnesota
Subject: Allosteric Regulation of Biological Function of Photoreceptor proteins

Major light-harvesting complex of photosystem II (LHCII) is a photoreceptor protein that regulates energy transfer and dissipation in response to rapid fluctuations of light intensity, directly affecting the efficiency of photosynthesis. In this presentation, I will describe an investigation combining molecular dynamics simulation and temperature-jump time-resolved IR spectroscopy to understand the mechanism of energy dissipation in LHCII. I will illustrate an allosteric regulation of the global protein conformational changes induced by a local conformational transition of random coils into α-helices due to changes of external temperature and acidity. The dynamic motions induce close contacts between the associated luteins (Lut) and photoactivated chlorophyll (Chl) chromophores to facilitate fluorescence quenching. In addition, I will discuss a multistate density functional theory designed to model photochemical and charge transfer processes.


Thursday, February 21st 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Gordon Smith, Assistant Professor, Department of Neuroscience, UMN
Subject: Development of large-scale networks in visual cortex.

Sensory perception requires the coordinated activity of tens of thousands of neurons, working together in large-scale networks. As developmental events define and constrain the ultimate capabilities of these networks, it is therefore essential to understand the mechanisms underlying their formation. This talk will present recent work showing that in the developing visual cortex, correlations in spontaneous neural activity define large-scale functional networks with precise local and long-range organization that span millimeters of cortical area. These early networks predict future stimulus-evoked activity well before it can be visually driven, suggesting they form a substrate for building a mature large-scale functional architecture.


Thursday, February 28th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Paul Jardine, Associate Professor, Department of Diagnostic and Biological Sciences, UMN
Subject: Using Model Systems to Drive Methods Development – The Tale of a Viral RNA

Model systems serve a critical role in the development of new research methodologies. By their nature, model systems are well defined and therefore present excellent opportunities to extend the resolution, range of application, and rigour of advanced biochemical and biophysical experimental techniques. Given that they are, by definition, some of the simplest living systems, viral model systems have been used to advance all areas of molecular biology and biophysics. Here, I will summarise the experimental history of one small component of a viral force generator nanomotor – the prohead RNA (pRNA) component of the bacteriophage phi29 DNA packaging machine – and illustrate how the study of this molecule has revealed fundamental insight into biological macromolecules. The study of pRNA has contributed to the development of experimental approaches that can be adapted to more complex systems in order to address more complex questions in biological systems.


Thursday, March 7th 2019
10:10 am:
Biophysics Seminar in 120 PAN
No speaker this week.

Thursday, March 21st 2019
10:10 am:
Biophysics Seminar in 120 PAN
Spring Break - No speaker this week.

Thursday, March 28th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jerome C. Mertz, College of Engineering, Boston University
Subject: Fast, volumetric imaging with microscopes

Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. Camera-based microscopes are typically hampered by the problem of out-of-focus background which undermines image contrast. This background must be reduced, or eliminated, to achieve volumetric imaging. Alternatively, scanning techniques such as confocal and multiphoton microscopy can provide high contrast and high speed, but their generalization to volumetric imaging requires an axial scanning mechanism, which, in general, drastically reduces speed. I will describe a variety of strategies we have developed to enable fast, high-contrast, volumetric imaging over large length scales. These strategies include targeted-illumination widefield microscopy, multi-z confocal microscopy and reverberation multiphoton microscopy. I will discuss the principles of these strategies and present experimental validations.

Faculty Host: Jochen Mueller

Thursday, April 11th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Mark Sanders, Program Director, University of Minnesota Imaging Centers
Subject: Frontiers in Imaging Technologies and Strategies for Researchers at University Imaging Centers

Core facilities are an essential resource in research institutes.The University Imaging Centers (UIC) has instrumentation, staffing expertise available for teaching, training and outreach in the imaging pipeline from experimental design through analysis. The UICs instrumentation list ranges from nano to mesoscales and includes electron microscopy; super-resolution; single and multi-photon confocal microscope systems; and high-content screening (HCS) systems. We have added tissue clearing, light sheet imaging and, coming soon, a mass spectrometry-based imaging platform in mid-2019. At the mesoscale, the UIC is equipped for in vivo small animal imaging providing investigators with bioluminescence, fluorescence, x-ray, µCT, µPET and ultrasound. How the UIC can help you would be our goal.


Thursday, April 18th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Erin Sheets, Associate Professor, University of Minnesota, Duluth, Department of Chemistry and Biochemistry
PLEASE NOTE THAT THE SEMINAR FOR THIS WEEK IS CANCELLED
Faculty Host: Elias Puchner

Thursday, April 25th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Siddarth Karuka, PhD student in Jochen Mueller’s lab, School of Physics and Astronomy
Subject:  Progress Report: Axial Super-Resolution with Two-Photon Microscopy

The Nuclear Envelope (NE) is a ~40 nm space enclosed by the Outer and Inner Nuclear Membrane (ONM and INM), that separates the nucleus from the cytoplasm. Although recent research has identified the NE as a critical signaling hub for a cell, it remains difficult to study with current fluorescence microscopy techniques that are limited to 50 nm axial resolution. To study systems like these, we have developed the dual color z-scan (DC Z-Scan) technique that can achieve axial resolution on the order of a nanometer. We first demonstrate the technique on a supported lipid bilayer, and then use it to measure the thickness of NE, distinguish proteins that reside on the ONM vs INM and study the translocation kinetics of these proteins.


Thursday, May 2nd 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Prof. Louis M. Mansky, Director, Institute for Molecular Virology, University of Minnesota
Subject: "Biophysical and Molecular Studies of Human Retroviral Assembly"

An underexplored aspect of human retrovirus replication are the steps involved in virus particle assembly. In particular the behavior of Gag movement to the plasma membrane, the engagement of particle budding sites, the molecular Gag-Gag interactions that create the immature Gag lattice, and subsequent particle biogenesis and morphogenesis remain poorly understood. Detailed comparative analysis of close relatives can be highly informative for gaining new insight into these steps in virus replication. Our interdisciplinary collaborative research team has made key observations regarding the differences in the pathways for Gag nucleation leading to punctum formation, as well as the nature of particle biogenesis also remain poorly understood aspects of the retrovirus assembly pathway, particularly among human immunodeficiency virus type 1 and its close relatives – i.e., human immunodeficiency virus type 2 (HIV-2) and human T-cell leukemia virus type 1 (HTLV-1). This lecture will discuss ongoing collaborative studies regarding 1) a comparative analysis of immature and mature virus particles and 2) an investigation of the nature of human retrovirus particle biogenesis. To date, our observations provide new insights into a highly significant and poorly understood aspect of the human retroviral life cycle, which has utility in informing intervention strategies.


Thursday, May 9th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Angel Mancebo, University of Minnesota
Subject: Mitigating phototoxicity in (super-resolution) fluorescence microscopy using precisely calibrated feedback illumination

Fluorescence microscopy is a powerful method for measuring spatial and dynamic information of specifically-labeled proteins in living cells. With the development of superresolution fluorescence microscopy methods diffraction-limited structures can be resolved and quantitative measurements of the spatio-temporal organization of proteins can be made. Because single-molecule localization-based superresolution microscopy methods rely on a spatio-temporally sparse distribution of fluorescent labels, long data acquisition times and high excitation powers are needed to localize a high fraction of molecules. Satisfying these two requirements results in a large amount of energy delivered to the cells by the lasers which is detrimental to cell health. The unfavorable implications are two-fold: unintended cell stress may compromise an experiment; and dying cells become autofluorescent making it impossible to detect single molecules. We developed a technique which makes use of a digital mirror array for accurately and precisely patterning the short-wavelength activation laser. This technique can be applied to any conventional or superresolution fluorescence microscopy experiment that requires spatial patterning as well as optogenetic experiments. We demonstrate this technique on budding yeast for confining the activation laser to only the plasma membrane where proteins with the plextrin homology domain are labeled with the photoactivatable protein mEos2. Patterned photoactivation mitigates cell death which improves single-molecule statistics by enabling longer acquisition times and an increase in the number of single-molecule localizations.


Thursday, May 16th 2019
10:10 am:
Biophysics Seminar in 120 PAN
There will be no seminar this week.

Thursday, September 5th 2019
10:10 am:
Biophysics Seminar in 120 PAN
There will be no seminar this week.

Thursday, September 12th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Chiranjib Banerjee, University of Minnesota
Subject: Quantitative super-resolution analysis of ULK1 oligomeric states and cellular localization

Autophagy is a complex biological process that degrades and recycles cellular components. The induction of autophagy involves complex formation and membrane association of various regulatory proteins. However, the underlying molecular processes at a nanoscopic length scale remain enigmatic. Here, we combine quantitative super-resolution microscopy with endogenous tagging to study the oligomeric states and subcellular localization of ULK1, the protein kinase that plays a central role in autophagy induction. Our results reveal with single-molecule sensitivity that ULK1 exists as a distribution of oligomers with up to 50 molecules in nutrient-rich condition. In the amino-acid deficient condition, however, ULK1 first forms dense oligomeric clusters with nearly 100 molecules. These clusters then transition to loosely bound spherical oligomeric structures with radii between 100 nm and 400 nm without any further increase in the number of ULK1 molecules. Co-localization of ULK1 with its interaction partner ATG13 confirms that the ULK1 oligomers are implicated in the autophagy initiation process. Therefore, a critical number of ULK1 molecules is required to initiate autophagy. The dense clusters are in close proximity to the ER while the larger loosely bound structures are located further away from the ER. Moreover, the larger spherical structures are also co-localized with the phagophore marker LC3B which suggests that ULK1 also participates in the late stage of autophagy as well. This single molecule image analysis demonstrates the dynamic changes of autophagy initiation proteins at an unprecedented level of resolution.


Thursday, September 19th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Chiranjib Banerjee, UMN, Puchner Lab
Subject: Quantitative super-resolution analysis of ULK1 oligomeric states and cellular localization

Autophagy is a complex biological process that degrades and recycles cellular components. The induction of autophagy involves complex formation and membrane association of various regulatory proteins. However, the underlying molecular processes at a nanoscopic length scale remain enigmatic. Here, we combine quantitative super-resolution microscopy with endogenous tagging to study the oligomeric states and subcellular localization of ULK1, the protein kinase that plays a central role in autophagy induction. Our results reveal with single-molecule sensitivity that ULK1 exists as a distribution of oligomers with up to 50 molecules in nutrient-rich condition. In the amino-acid deficient condition, however, ULK1 first forms dense oligomeric clusters with nearly 100 molecules. These clusters then transition to loosely bound spherical oligomeric structures with radii between 100 nm and 400 nm without any further increase in the number of ULK1 molecules. Co-localization of ULK1 with its interaction partner ATG13 confirms that the ULK1 oligomers are implicated in the autophagy initiation process. Therefore, a critical number of ULK1 molecules is required to initiate autophagy. The dense clusters are in close proximity to the ER while the larger loosely bound structures are located further away from the ER. Moreover, the larger spherical structures are also co-localized with the phagophore marker LC3B which suggests that ULK1 also participates in the late stage of autophagy as well. This single molecule image analysis demonstrates the dynamic changes of autophagy initiation proteins at an unprecedented level of resolution.

Faculty Host: Elias Puchner

Thursday, September 26th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jan Huisken, Investigator, Morgridge Medical Engineering and Visiting Professor, Integrative Biology
Subject: Putting advanced light sheet fluorescence microscopy in the hands of biologists with modular and portable instruments

Discoveries in biology often depend on cutting edge technologies such as microscopy. Biologists mostly rely on commercial microscopes, which are technically mature but often outdated and not well-tailored to the individual experiment’s requirements. An example is light sheet microscopy (SPIM), which has changed the field of fluorescence imaging substantially by offering a versatile technique to obtain optical sectioning in large specimens with high speed and minimal phototoxicity. The ability to custom design a SPIM instrument around a sample has empowered many research labs to do experiments that have been impossible with commercial instruments. I will give a few examples from our lab to illustrate how biologists use the technology to study morphogenesis and function in living organisms, primarily in zebrafish.
Unfortunately, only physics and engineering labs have been able to custom design such an instrument to enable demanding biological applications. We have addressed this issue by developing the Flamingo (www.involv3d.org/flamingo), a modular, shareable light sheet microscope suited to a new model of scientific collaboration. Each microscope is customized for a given application, equipped to travel from lab to lab and to provide widespread access to advanced microscopy. With the Flamingo traveling back and forth between our lab and the partner labs, we hope to iteratively refine the technology and constantly improve the instrument. Driven by the ever-growing number of applications for light sheet microscopy, we will expand the capabilities of our Flamingo framework and thereby advance the optics as well as the biological experiments that our instrument enables. Most importantly, we address a major drawback of current collaboration models and imaging facilities: typically, the biologists and their samples need to travel to the microscope, inducing stress on the biological system. In our approach, the biologists stay in their lab where they can best perform the experiments, strengthening collaborations between the engineers and the biologists, while ensuring the experiment’s quality and reproducibility at the highest standards.

Suggested Literature
Power, Huisken, Nature Methods, 14, 360 (2017)
Berndt, Shah, Brugues, Huisken, Nature Communications, 9, 5025 (2018)
Daetwyler, Günther, Modes, Harrington, Huisken, Development 146, dev173757 (2019)


Thursday, October 3rd 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Ryan Marshall, UMN, Noireaux Lab
Subject: An E. coli cell-free transcription-translation system: modeling gene expression and characterizing CRISPR elements and gene circuits

Cell-free transcription-translation systems are versatile tools for rapid prototyping and characterization of biological systems and processes. Proteins can be expressed and measured in a matter of hours, whereas in vivo experiments often take days to weeks because they require protein purification or live cell transformations and cultures. TXTL systems, however, are still lacking in simple models that quantitatively describe the behavior of reactions. Here, we present an model of the all E. coli TXTL system using ordinary differential equations, encompassing the limited concentrations of transcription and translation machineries, capturing the linear and saturated regime of gene expression. Many biochemical constants are determined through experimental assays. We then show how this TXTL system was used to characterize CRISPR technologies. CRISPR-Cas systems have huge potential to be used as tools for genome engineering, as well as gene silencing and regulation. We characterize a set of sgRNAs, CRISPR nucleases, anti-CRISPR proteins, and determine protospacer-adjacent motifs. Finally, we use the TXTL system to execute gene circuits, including an IFFL and an integral controller.


Thursday, October 10th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Asset Khakimzhan, UMN, Noireaux Lab
Subject: CRISPR in TXTL: Statistical Mechanics of CRISPR Target Recognition

CRISPR is without a doubt the most hyped acronym of modern molecular biology and not without a reason. CRISPR is an easy-to-use tool for editing, activating, and silencing genes in a broad range of biological systems. Improving our understanding of the rules that CRISPR systems follow could help expanding its scope of applications. Recently, it was shown that we can rapidly interrogate CRISPR systems using TXTL, an all E. Coli cell-free transcription-translation system, and achieve similar results to in-vivo experiments. TXTL provides easier access to characterizing the molecular mechanisms related to CRISPR. In this seminar, I will discuss our findings in CRISPR target recognition and provide a statistical mechanical model of how CRISPR systems decide on making DNA cuts. I will explain why TXTL is better suited for measuring CRISPR targeting mechanisms and how knowing the details of CRISPR targeting might be useful for in-vivo experiments. Finally, I will show how understanding the targeting rules allows us to trick nucleating CRISPR-Cas9 systems into activating or inhibiting a gene without having to mutate the Cas9 protein.


Thursday, October 17th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jared Hennen, UMN, Mueller Lab
Subject: Protein mobility and local volume fluctuations identify membrane binding of proteins within the sub-resolution structure of the nuclear envelope

The nuclear envelope (NE) consists of two concentric nuclear membranes that surrounds the nucleus from the cytoplasm in eukaryotic cells. The two nuclear membranes are separated by a thin fluid layer, referred to as the lumen, which is densely packed with proteins. These proteins are implicated in important cellular processes, such as mechano-regulated gene expression. The molecular details of these regulation processes, which typically require the formation of protein complexes, are not well understood due to a lack of techniques for the physical characterization of protein assembly in the NE of living cells. While recent progress has been made using fluorescence fluctuation spectroscopy (FFS) to quantify the assembly states of NE proteins in their native environment, monitoring the interaction of proteins with the membrane during assembly remains an unsolved problem. This is a significant shortcoming because membrane binding is often associated with conformational changes in proteins that are critical to cellular signaling pathways. Particularly vexing for studying membrane association is the close proximity of the nuclear membranes, which are only separated by the approximately 40 nm thick lumen. Thus, optical resolution is insufficient to directly distinguish luminal and membrane-bound NE proteins by fluorescence imaging methods.

To overcome this obstacle, we examined protein mobility within the NE by FFS methods. While membrane association has been detected using mobility in other regions of the cell, the confined spatial structure of the NE requires us to check the validity of the Stokes-Einstein relation for luminal proteins. In addition, we explore the temperature-dependent mobility of soluble and membrane-bound proteins in the NE as a potential marker for differentiating these populations. Finally, we look at the undulations of the nuclear membranes, which introduce local volume changes. This process is detected by FFS as an additional fluctuation signal for luminal proteins, but is absent for membrane-bound proteins. We harness this difference to provide a second, independent tool for identifying membrane-bound NE proteins. These new techniques are then applied to investigate the membrane association of two proteins native to the NE: SUN2, a constituent protein of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, and the AAA+ ATPase torsinA.


Thursday, October 24th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Rayna Addabbo, UMN
Subject: The complementary role of co- and post-translational events in de novo protein synthesis

While required for life, protein folding is poorly understood, especially in the context of the complex environment of the cell. Here, I report recent findings on the relationship between co- and post-translational protein folding and protein aggregation. I show that the ribosome promotes cotranslational nascent-protein solubility, thus supporting cotranslational folding even in the absence of molecular chaperones. The ribosome alone, however, does not guarantee quantitative formation of the native state. Upon completion of protein biosynthesis, there is a crucial irreversible post-translational kinetic partitioning between protein folding and protein aggregation. De novo-synthesized proteins only attain their native state if the rates of soluble and insoluble aggregate formation upon release from the ribosome are slow relative to intramolecular folding. Mutational analysis demonstrates that the above post-translational kinetic partitioning events are more sensitive to amino-acid sequence than the native fold, suggesting post-translational kinetic partitioning is a source of significant evolutionary pressure.


Thursday, October 31st 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: TBA

Thursday, November 7th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Comert Kural, Ohio State University
Subject: Curvature Generation by Endocytic Clathrin Coats: Super-resolution Imaging Resolves Forty Years of Controversy

Clathrin-mediated endocytosis is the most extensively studied internalization mechanism of membrane lipids and proteins from the cell surface. Over the past decades, a multitude of biophysical and biochemical methodologies have been employed to elucidate structural and dynamic properties of endocytic clathrin coats. However, fundamental aspects of clathrin-mediated endocytosis remain controversial due to the lack of experimental approaches that allow correlation of ultra-structural and dynamic properties of clathrin-coated structures. Using electron micrographs, it was originally proposed that clathrin initially grows into a flat array (i.e., clathrin plaques) on the plasma membrane prior to transitioning into a curved coat. Flat-to-curved transition of clathrin coats during endocytic vesicle formation was rejected by others as it requires a substantial structural rearrangement, which is energetically unfavorable. As an alternative, it was suggested that curved clathrin-coated structures form gradually without a major structural rearrangement. In this study, we used structured illumination microscopy in the total internal reflection mode to monitor curvature formation by clathrin coats during assembly of individual endocytic complexes within cultured cells and tissues of developing metazoan organisms. Our analyses very clearly demonstrate that endocytic clathrin coats acquire curvature without a major flat-to-curved transition that requires an extensive reorganization of the clathrin lattice. Altogether, our results signify the importance of employing methodologies comprising high resolution in both spatial and temporal dimensions for constructing dynamic models.

Faculty Host: Jochen Mueller

Thursday, November 14th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: TBA

Thursday, November 21st 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: TBA

Thursday, November 28th 2019
10:10 am:
Biophysics Seminar in 120 PAN
No seminar - Thanksgiving Break

Thursday, December 5th 2019
10:10 am:
Biophysics Seminar in 120 PAN
There will be no seminar this week.

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