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Capturing the Dynamic, Heterogeneous Response of Microbes to their Environment in the Human Microbiome
4a
Saturday, February 27, 2016
24-Subg Cotranslational Protein Folding Gunnar von Heijne. Dept Biochem/Biophys, Stockholm University, Stockholm, Sweden. We have developed a technique based on the use of so-called translational arrest peptides to measure forces acting on a nascent chain during cotranslational processes such as membrane translocation and folding. In contrast to hard-core biophysical methods, the technique can be used both in vitro and in vivo. Recent results on cotranslational folding of cytoplasmic proteins will be presented. 25-Subg Some Cell Behavior is Encoded in Proteome Physics Ken Dill. Laufer Center for Physical & Quantitative Biology, Stony Brook University, Stony Brook, NY, USA. More than half the biomass of a bacterial cell is protein. So, some cell behaviors may arise from protein physics -- folding, aggregation, oxidation, and diffusional transport, for example. We develop simple physical models for how protein physics affects cell growth. Cells are sensitive to temperature. We believe this is because cell’s live near protein denaturation temperatures. Cells grow slowly in high salts. We believe this is because osmotic shrinkage of the cell crowds proteins, slowing their diffusional transport. A cell’s proteome oxidizes with age. We believe this results in the electrostatic unfolding of high-charge proteins. Reasoning with simple models also gives insights into a cell’s energy balance, replication speed, and others.
Subgroup: Nanoscale Biophysics 26-Subg Nanoscope Study of Chromatin Structure and Process in Mammalian Cells Yujie Sun. BIOPIC, Peking University, Beijing, China. DNA replication is tightly regulated in time and space. Previous studies have suggested that there are nearly 300-500 thousand replication origins in a mammalian cell. However, during DNA replication, only 50,000 replication origins are fired. The mechanims of replication origin activation has been elusive. Using super-resolution microscopy, we focus on the structural features of chromatin DNA and propose they may be key factors to define the efficiency of replication origins. This study promotes the concept that chromatin structure is an important epigenetic code that regulates functions of chromatin DNA. 27-Subg Capturing the Dynamic, Heterogeneous Response of Microbes to their Environment in the Human Microbiome Julie Biteen. University of Michigan, Ann Arbor, MI, USA. It has long been recognized that microorganisms play a central role in our lives. Excitingly, new biophysical methods like super-resolution imaging are letting us see inside cells, and we can understand the complexity that leads to bacterial subcellular function with increasing detail (Tuson and Biteen, 2015). Still, most bacteria cells do not live alone in petri dishes. Rather, the majority are members of microbial communities that live both in and on us, and profoundly influence our well-being. Thus, we need to understand the unique features of individual species that give rise to population-level observations, yet it is no longer sufficient for us to study cells in isolation. Rather, interactions between a cell and its environment are key factors in understanding the microbiome function and mechanism. There exists a fundamental knowledge gap between the molecular-scale understanding of isolated proteins in bacteria cells which can be determined from single-cell measurements and the cell- and community-level knowledge gleaned from discovery-based biochemical approaches. We have been bridging this gap by using single-molecule imaging in living cells to capture the dynamic response of enzymes in human gut microbes to their surroundings in real time on the nanometer scale. In particular, we are measuring the molecular-level response of key starch utilization proteins involved in glycan catabolism by a prominent human gut bacterium Bacteroides thetaiotaomicron (Karunatilaka et al., 2015). Overall, by developing a mechanistic framework in which to understand how individual species respond to environmental factors such as glycan concentration, we will understand the complex function of a microbiome. 28-Subg Converging and Correlative Technologies for Optical Nanoscopy Alberto Diaspro1,2, Paolo Bianchini1, Claudio Canale1, Francesca Cella Zanacchi1, Marta Duocastella1, Luca Lanzano`1, Nirmal Mazumder1, Colin Sheppard1, Giuseppe Vicidomini1. 1 Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of Physics, University of Genoa, Genova, Italy.
The sentence ‘‘Microscopy has become nanoscopy’’, reported in the Nobel Prize 2014 statement, updates ‘‘microscopium nominare libuit’’ referred to the Galileo Galilei’s optical microscope. So far, optical nanoscopy refers to those methods that in the last 25 years (circa) have been developed to crumble the diffraction limit by two far-field principles that lead to fluorescence-based microscopy with a spatial resolution that is further the Abbe’s limit. These methods have been referred as ‘‘super-resolved ensemble fluorophore microscopy’’ and as ‘‘superresolved single fluorophore microscopy’’ also known as targeted and stochastic read out methods, respectively [Diaspro, A. 2014. Il Nuovo Saggiatore]. We will discuss converging and correlative techniques linked to nanoscopy, a sort of roadmap for developments [Eggeling C. et al. 2015 QRB; Hell, S.W. et al. 2015. J.Phys.D; Li, D. et al. 2015. Science]. The main focus is related on imaging techniques that permit direct measurements of the live-cell molecular dynamics at the nanometer scale including the study of thick biological samples. An important list of converging technologies is currently under development, for example: recent advances in camera technology and real-time image processing have led to substantially improved time resolution, RESOLFT nanoscopy and the utilization of temporal information for decoding spatial information allowed super resolution at reduced beam intensities, the advent of new fluorescent molecules are providing better quantitative abilities, original image deconvolution approaches for noise removal, in vivo imaging, new lenses and beam shapers. What about creating an optical microscope capable to look at living cells with the resolution of an electron microscope? At the moment correlative approaches coupling optical super resolved methods with electron and scanning probe microscopes are providing interesting developments that will be outlined [Chacko, J.V. et al. 2013. Cytoskeleton; Monserrate, et al. 2013. ChemPhysChem; Viero G. et al. 2015 J.Cell.Biol.]. 29-Subg Seeing Single Molecules, from Early Spectroscopy in Solids, to SuperResolution Microscopy, to 3D Dynamics of Biomolecules in Cells W.E. Moerner. Chemistry, Stanford University, Stanford, CA, USA. More than 25 years ago, low temperature experiments aimed at establishing the ultimate limits to optical storage in solids led to the first optical detection and spectroscopy of a single molecule in the condensed phase. At this unexplored ultimate limit, many surprises occurred where single molecules showed both spontaneous changes (blinking) and light-driven control of emission, properties that were also observed in 1997 at room temperature with single green fluorescent protein variants. In 2006, PALM and subsequent approaches showed that the optical diffraction limit of ~200 nm can be circumvented with single molecules to achieve super-resolution fluorescence microscopy with relatively nonperturbative visible light. Super-resolution microscopy has opened up a new frontier in which biological structures and behavior can be observed in fixed and live cells with resolutions down to 20-40 nm and below. Current methods development research addresses ways to extract more information from each single molecule such as 3D position and orientation, and both of these can be obtained by proper point-spread function engineering of a wide-field microscope. It is worth noting that in spite of all the current focus on super-resolution, even in the ‘‘conventional’’ low concentration, single-molecule tracking regime where the motions of individual biomolecules are recorded rather than the shapes of extended structures, much can still be learned about biological processes. For example, my laboratory has explored the motions of single Smoothened proteins in the primary cilium, providing evidence of binding sites at the ciliary base whose affinity is modulated by Hedgehog pathway activation. Using 3D precision tracking at high speed, correlations in the motions of pairs of DNA loci in the yeast nucleus highlight the complexity of the nuclear environment through the appearance of subdiffusive motion.
Subgroup: Membrane Structure and Assembly 30-Subg Membrane Domains on the Sub-Nanometer Scale Georg Pabst. University of Graz, Graz, Austria. The existence of membrane domains/rafts is highly controversial in live cells, but well-established in lipid-only mimetics of mammalian plasma membranes. We apply in-situ small angle x-ray/neutron scattering experiments in combination with computational simulations to determine membrane structural properties of coexisting liquid-ordered (Lo)/liquid disordered (Ld) domains at high resolution, including their elastic moduli and fundamental interactions across the aqueous phase. Thereby we gain detailed insight into the physics pertaining to membrane domains. I will highlight our recent results on macroscopic and nanoscopic domains, including structural changes upon melting or decreasing domain size, domain elasticity and interactions, ion-specific effects, protein
Saturday, February 27, 2016
24-Subg Cotranslational Protein Folding Gunnar von Heijne. Dept Biochem/Biophys, Stockholm University, Stockholm, Sweden. We have developed a technique based on the use of so-called translational arrest peptides to measure forces acting on a nascent chain during cotranslational processes such as membrane translocation and folding. In contrast to hard-core biophysical methods, the technique can be used both in vitro and in vivo. Recent results on cotranslational folding of cytoplasmic proteins will be presented. 25-Subg Some Cell Behavior is Encoded in Proteome Physics Ken Dill. Laufer Center for Physical & Quantitative Biology, Stony Brook University, Stony Brook, NY, USA. More than half the biomass of a bacterial cell is protein. So, some cell behaviors may arise from protein physics -- folding, aggregation, oxidation, and diffusional transport, for example. We develop simple physical models for how protein physics affects cell growth. Cells are sensitive to temperature. We believe this is because cell’s live near protein denaturation temperatures. Cells grow slowly in high salts. We believe this is because osmotic shrinkage of the cell crowds proteins, slowing their diffusional transport. A cell’s proteome oxidizes with age. We believe this results in the electrostatic unfolding of high-charge proteins. Reasoning with simple models also gives insights into a cell’s energy balance, replication speed, and others.
Subgroup: Nanoscale Biophysics 26-Subg Nanoscope Study of Chromatin Structure and Process in Mammalian Cells Yujie Sun. BIOPIC, Peking University, Beijing, China. DNA replication is tightly regulated in time and space. Previous studies have suggested that there are nearly 300-500 thousand replication origins in a mammalian cell. However, during DNA replication, only 50,000 replication origins are fired. The mechanims of replication origin activation has been elusive. Using super-resolution microscopy, we focus on the structural features of chromatin DNA and propose they may be key factors to define the efficiency of replication origins. This study promotes the concept that chromatin structure is an important epigenetic code that regulates functions of chromatin DNA. 27-Subg Capturing the Dynamic, Heterogeneous Response of Microbes to their Environment in the Human Microbiome Julie Biteen. University of Michigan, Ann Arbor, MI, USA. It has long been recognized that microorganisms play a central role in our lives. Excitingly, new biophysical methods like super-resolution imaging are letting us see inside cells, and we can understand the complexity that leads to bacterial subcellular function with increasing detail (Tuson and Biteen, 2015). Still, most bacteria cells do not live alone in petri dishes. Rather, the majority are members of microbial communities that live both in and on us, and profoundly influence our well-being. Thus, we need to understand the unique features of individual species that give rise to population-level observations, yet it is no longer sufficient for us to study cells in isolation. Rather, interactions between a cell and its environment are key factors in understanding the microbiome function and mechanism. There exists a fundamental knowledge gap between the molecular-scale understanding of isolated proteins in bacteria cells which can be determined from single-cell measurements and the cell- and community-level knowledge gleaned from discovery-based biochemical approaches. We have been bridging this gap by using single-molecule imaging in living cells to capture the dynamic response of enzymes in human gut microbes to their surroundings in real time on the nanometer scale. In particular, we are measuring the molecular-level response of key starch utilization proteins involved in glycan catabolism by a prominent human gut bacterium Bacteroides thetaiotaomicron (Karunatilaka et al., 2015). Overall, by developing a mechanistic framework in which to understand how individual species respond to environmental factors such as glycan concentration, we will understand the complex function of a microbiome. 28-Subg Converging and Correlative Technologies for Optical Nanoscopy Alberto Diaspro1,2, Paolo Bianchini1, Claudio Canale1, Francesca Cella Zanacchi1, Marta Duocastella1, Luca Lanzano`1, Nirmal Mazumder1, Colin Sheppard1, Giuseppe Vicidomini1. 1 Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of Physics, University of Genoa, Genova, Italy.
The sentence ‘‘Microscopy has become nanoscopy’’, reported in the Nobel Prize 2014 statement, updates ‘‘microscopium nominare libuit’’ referred to the Galileo Galilei’s optical microscope. So far, optical nanoscopy refers to those methods that in the last 25 years (circa) have been developed to crumble the diffraction limit by two far-field principles that lead to fluorescence-based microscopy with a spatial resolution that is further the Abbe’s limit. These methods have been referred as ‘‘super-resolved ensemble fluorophore microscopy’’ and as ‘‘superresolved single fluorophore microscopy’’ also known as targeted and stochastic read out methods, respectively [Diaspro, A. 2014. Il Nuovo Saggiatore]. We will discuss converging and correlative techniques linked to nanoscopy, a sort of roadmap for developments [Eggeling C. et al. 2015 QRB; Hell, S.W. et al. 2015. J.Phys.D; Li, D. et al. 2015. Science]. The main focus is related on imaging techniques that permit direct measurements of the live-cell molecular dynamics at the nanometer scale including the study of thick biological samples. An important list of converging technologies is currently under development, for example: recent advances in camera technology and real-time image processing have led to substantially improved time resolution, RESOLFT nanoscopy and the utilization of temporal information for decoding spatial information allowed super resolution at reduced beam intensities, the advent of new fluorescent molecules are providing better quantitative abilities, original image deconvolution approaches for noise removal, in vivo imaging, new lenses and beam shapers. What about creating an optical microscope capable to look at living cells with the resolution of an electron microscope? At the moment correlative approaches coupling optical super resolved methods with electron and scanning probe microscopes are providing interesting developments that will be outlined [Chacko, J.V. et al. 2013. Cytoskeleton; Monserrate, et al. 2013. ChemPhysChem; Viero G. et al. 2015 J.Cell.Biol.]. 29-Subg Seeing Single Molecules, from Early Spectroscopy in Solids, to SuperResolution Microscopy, to 3D Dynamics of Biomolecules in Cells W.E. Moerner. Chemistry, Stanford University, Stanford, CA, USA. More than 25 years ago, low temperature experiments aimed at establishing the ultimate limits to optical storage in solids led to the first optical detection and spectroscopy of a single molecule in the condensed phase. At this unexplored ultimate limit, many surprises occurred where single molecules showed both spontaneous changes (blinking) and light-driven control of emission, properties that were also observed in 1997 at room temperature with single green fluorescent protein variants. In 2006, PALM and subsequent approaches showed that the optical diffraction limit of ~200 nm can be circumvented with single molecules to achieve super-resolution fluorescence microscopy with relatively nonperturbative visible light. Super-resolution microscopy has opened up a new frontier in which biological structures and behavior can be observed in fixed and live cells with resolutions down to 20-40 nm and below. Current methods development research addresses ways to extract more information from each single molecule such as 3D position and orientation, and both of these can be obtained by proper point-spread function engineering of a wide-field microscope. It is worth noting that in spite of all the current focus on super-resolution, even in the ‘‘conventional’’ low concentration, single-molecule tracking regime where the motions of individual biomolecules are recorded rather than the shapes of extended structures, much can still be learned about biological processes. For example, my laboratory has explored the motions of single Smoothened proteins in the primary cilium, providing evidence of binding sites at the ciliary base whose affinity is modulated by Hedgehog pathway activation. Using 3D precision tracking at high speed, correlations in the motions of pairs of DNA loci in the yeast nucleus highlight the complexity of the nuclear environment through the appearance of subdiffusive motion.
Subgroup: Membrane Structure and Assembly 30-Subg Membrane Domains on the Sub-Nanometer Scale Georg Pabst. University of Graz, Graz, Austria. The existence of membrane domains/rafts is highly controversial in live cells, but well-established in lipid-only mimetics of mammalian plasma membranes. We apply in-situ small angle x-ray/neutron scattering experiments in combination with computational simulations to determine membrane structural properties of coexisting liquid-ordered (Lo)/liquid disordered (Ld) domains at high resolution, including their elastic moduli and fundamental interactions across the aqueous phase. Thereby we gain detailed insight into the physics pertaining to membrane domains. I will highlight our recent results on macroscopic and nanoscopic domains, including structural changes upon melting or decreasing domain size, domain elasticity and interactions, ion-specific effects, protein