Biophysics Seminar

All future

Thursday, September 27th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Keehun Kim, graduate student in Sivaraj Sivaramakrishnan’s lab, Department of Genetics, Cell Biology, and Development, University of Minnesota
Subject: A cell-free FRET-based assay for profiling the intrinsic efficacy of GPCR ligands

G protein coupled receptors (GPCRs) are 7-helix transmembrane receptors which regulate diverse intracellular signaling cascades in response to extracellular stimuli. Due to their crucial role in modulating most human physiological processes, roughly 25% of the pharmaceutical in the current market target GPCRs. The intrinsic efficacy of GPCR ligands is an essential parameter in operational models that convert ligand binding to downstream response. Measurement of intrinsic efficacy of GPCR ligands has eluded researchers, given the diversity of factors that can modulate downstream signaling, including expression levels of GPCR, G protein-dependent and independent effectors, regulatory factors such GRKs, RGS, and post-translational modifications. While ligand efficacy can be directly measured from G protein activation rates, such assays either require significant amounts of purified GPCR and G protein or are limited by the heterogeneity inherent in crude membrane preparations. We have previously used FRET based SPASM sensors to measure interactions between the full-length GPCR and the C-terminus of the α5-helix of distinct Gα subunits. We have demonstrated the utility of these sensors in live cells to probe the stabilization of G protein-selective receptor conformations by distinct GPCR ligands. While the SPASM GPCR sensors robustly report ligand-dependent G protein selection, the live cell FRET measurements are limited by constraints on sensor expression levels and ligand-stimulation times. Here, we present a cell derived assay for SPASM GPCR sensors that provides highly reproducible FRET measurements with minimal sensitivity to sensor expression levels and ligand-stimulation times. Importantly, our cell-free assay provides a scalable and stable reagent for screening GPCR ligands. Our findings for the b2 adrenergic receptor show that the GPCR-Gα C-terminus interaction is sufficient to recapitulate the intrinsic efficacy as measured from G protein activation rates. Taken together, our cell-free assay provides a scalable FRET-based readout for the intrinsic efficacy of GPCR ligands, while reinforcing the structural mechanisms underlying G protein activation.

Thursday, October 4th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Jon Garamella, graduate student in Vincent Noireaux’s lab, School of Physics and Astronomy, University of Minnesota
Subject: Synthetic cell prototyping using a cell-free transcription-translation system (TXTL)

Cell-free Transcription-translation (TXTL) systems can be used in synthetic cell engineering to reconstitute cellular processes and functions. The bottom-up construction of cell-sized compartments programmed with DNA that are capable of sensing their chemical and physical environment remains challenging. Here, we develop techniques to prototype synthetic cells with passive and active membrane functionality. We construct liposomes programmed to intake nutrients from the surroundings and respond to external, mechanical stimuli using alpha-hemolysin and the mechanosensitive channel of large conductance, respectively. High-throughput techniques to test the viability of membrane proteins are also described.

Thursday, October 11th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Dushyant Mehra, graduate student in the biomedical engineering department at the Mayo Clinic
Subject: Mapping and Manipulation of DNA Architecture in Vivo

Genome organization helps regulate gene expression and is an important driver in processes associated with cell development and cell differentiation. Enhancer and insulator complexes that dynamically form play central roles in governing transcriptional dynamics of genes that are associated processes such as cell fate determination and alterations in organization can often lead to disease. Current crosslinking based methods such that map DNA conformations by measuring crosslinking frequency often don’t account for changes in chromatin architecture across different cell states. Recent research suggests that analyzing speed of DNA interaction and not just contact frequency is important in finding DNA contacts that govern gene expression and DNA contacts that important in governing gene expression may not be picked up by current crosslinking based mapping techniques. Dynamic imaging based methods have been used to track dynamics of DNA overtime but are often difficult to apply due conventional microscopy limits which makes it difficult to track dynamics of clustered enhancers and insulators. By employing CRISPR-dCas9 DNA binding domains and super resolution photo-localization microscopy, we aim to optically map chromatin architecture and track dynamics of DNA during a variety of cell states and by tethering sections of DNA together, we are able to understand how these contacts affect affects gene expression. Together, we are able to develop a toolkit to understand the role that chromatin architecture plays in transcription during different cellular processes and cell states.

Thursday, October 18th 2018
3:35 pm:
Biophysics Seminar in B50 Tate 
Speaker: Terry Hwa, Department of Physics, University of California at San Diego Physics and Astronomy Colloquium
(Note: Terry Hwa will not give a Biophysics Seminar but just a colloquium)

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 1st 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Douglas G. Mashek, Professor, Department of Biochemistry, Molecular Biology and Biophysics and Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, University of Minnesota
Subject: The fascinating world of lipid droplet biology

Lipid droplets are recognized to be the primary storage form of energy in most cell types. While this important function is critical to supply cells or organisms with energy during times of nutrient deprivation, their role beyond energy storage has largely been unexplored. Our laboratory has focused on elucidating the mechanisms through which lipid droplets communicate within cells to coordinate energy storage with cell signaling and metabolism. Our work on hepatic lipid droplets has identified important functions for these dynamic organelles in regulating lipid and glucose metabolism, hormone signaling and cell proliferation. Specifically, data will be presented showing that lipid droplet catabolism promotes signaling networks to control mitochondrial biogenesis and function. Moreover, recent insights into the mechanisms through which lipid droplets are catabolized via autophagic pathways will also be discussed. In summary, this presentation will highlight novel roles for lipid droplets and their metabolism in cellular and organismal biology and their importance in human health.

Thursday, November 8th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Isaac Angert, graduate student in Jochen Mueller’s lab, School of Physics and Astronomy, University of Minnesota

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.

Thursday, November 29th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Santosh Adhikari, graduate student in Elias Puchner's lab, School of Physics and Astronomy, University of Minnesota

Thursday, December 6th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Steven Vogel, NIH/NIAAA

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