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Catalytic Intermediates of Membrane Fission
158a
Monday, February 13, 2017
Symposium: Biophysics of lncRNA 777-Symp Dynamic Temporal Control of Signaling Activated Gene Regulation Gregor Neuert. Vanderbilt University, Nashville, TN, USA. Many signal transduction and gene regulatory pathways are highly dynamic resulting in temporally varying activity of signaling proteins and time varying gene expression profiles. These time varying signaling profiles are important as these can direct different cellular phenotypes. Currently, our biophysical understanding of these temporal signaling profiles is very rudimentary and relies on the genetic manipulation of specific proteins or drug treatment regimes. Although protein numbers are often manipulated over a wide dynamic range using an inducible promoter, the activity of these signaling proteins may only be manipulated by mutations or drugs. One drawback of this approach is that genetic protein modification may result in a significant interference with the function of the cell, which may cause lethal phenotypes. Another drawback is that drugs are often not available for proteins of interest or have considerable off-target effect. In order to avoid these limitations, we developed a time varying perturbation approach that utilizes time varying ligand profiles to investigate the temporal properties of signaling and gene regulatory pathways, without genetic manipulation or drug treatments. To demonstrate the feasibility of this approach, we chose to interrogate an evolutionary conserved stress response pathway in yeast S. Cerevisiae, which enables to manipulate the intensity, duration and temporal activity of the signal transduction pathway in single cells. We are able to quantify signal transduction activation, signal transduction saturation and gene expression activation thresholds at high precision and demonstrate that the signaling dynamics is proportional to the first time derivative of the external perturbation profile. Because this approach is independent of the biological pathway or organism, it presents a general methodology to interrogate and control signaling and gene expression pathways non-invasively without the need for genetic or drug perturbations. 778-Symp The Ground-State of Promoter Directionality Revealed by a Functional Evolutionary Approach and Deep Learning Modeling Stirling Churchman. Department of Genetics, Harvard Medical School, Boston, MA, USA. Although the RNA polymerase (Pol) II machinery inherently initiates transcription in one direction, promoter regions are often ‘‘bidirectional’’ in vivo, giving rise to divergent RNA transcripts, many of which are noncoding and highly unstable. Here, we use a functional evolutionary approach to address whether bidirectional promoter regions are a mechanistic consequence of Pol II transcription or serve an evolved biological function. This involves nascent transcript mapping in S. cerevisiae strains containing large segments of foreign, and hence evolutionarily irrelevant, yeast DNA. A deep learning based model allows the unbiased identification of transcription start sites that emerge in the foreign DNA. Promoter regions in foreign species environments lose the directionality they have in their native species, indicating that DNA sequences and proteins co-evolve to promote directional transcription. Furthermore, fortuitous promoters emerge frequently in foreign DNA, and these produce equal transcription in both directions. Thus, without evolutionary pressure, the transcriptional ground state of promoter regions is intrinsically bidirectional. These results indicate that promoter regions are intrinsically bidirectional and are shaped by evolution to bias transcription of coding transcripts while suppressing non-coding transcriptional noise. 779-Symp How a lncRNA Shapes Chromatin Structure to Control Gene Expression Mitchell Guttman. Caltech, Pasadena, CA, USA. Mammalian genomes encode many thousands of long non-coding RNAs (lncRNAs) that play important roles in diverse biological processes. As a class, lncRNAs are generally enriched in the nucleus and specifically within the chromatin-associated fraction. Here, we will discuss the evidence that many nuclear-retained lncRNAs can interact with various chromatin regulatory proteins and recruit them to specific sites on DNA to regulate gene expression and shape three-dimensional nuclear organization. Specifically, we will discuss emerging mechanistic insights derived from the Xist lncRNA, a paradigm for lncRNA-mediated gene regulation, into how lncRNAs regulate gene expression by localizing to genomic target sites, recruiting regulatory complexes, and reshaping nuclear organization.
780-Symp Structure vs. Function: A Quantitative Analysis of Chromosome Architecture Luca Giorgetti. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. In animals, transcriptional regulation by distal genetic elements such as enhancers occurs in the context of the complex three-dimensional architecture of chromosomes. Experiments based on chromosome conformation capture such as Hi-C have shown that mammalian chromosomes are folded into a rich hierarchy of structural layers. At the core of this hierarchy, topologically associating domains (TADs) and their substructures appear to constrain and fine-tune the three-dimensional interactions of regulatory sequences, thus contributing to the establishment and maintenance of the correct gene expression patterns. We use a combination of quantitative experiments, genomic engineering and physical modeling to dissect the biophysical mechanisms by which chromosome structure and dynamics control transcriptional regulation.
Symposium: Catalyzed Membrane Fusion and Fission 781-Symp Know when to Hold ‘Em, Know when to Fold ‘Em; SM Proteins as Templates for SNARE Assembly Frederick Hughson. Department of Molecular Biology, Princeton University, Princeton, NJ, USA. A major focus of our research is the machinery that guides the fusion of vesicles during intracellular transport. These vesicles deliver their cargo via membrane fusion reactions executed by membrane-bridging SNARE complexes. The formation of SNARE complexes generally requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. Consequently, we hypothesize that SNARE complex assembly reactions in the cell are orchestrated by a set of ‘topologically aware’ chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing fourhelix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with - and sometimes as integral subunits of - the MTCs. Therefore, the goal of our work is to achieve an improved structural and mechanistic understanding of MTC and SM function in the assembly of fusogenic SNARE complexes. My presentation will focus on the SM proteins, and on our X-ray crystallographic and biochemical efforts to understand how they catalyze four-helix bundle formation. 782-Symp Catalytic Intermediates of Membrane Fission Vadim A. Frolov1, Pavel V. Bashkirov2, Anna V. Shnyrova1. 1 Biofisica Institute, Univ. Basque Country, Leioa, Spain, 2Federal Research and Clinical Centre of Physical-Chemical Medicine, Moscow, Russian Federation. Fusion and fission of cellular membranes entail localized transformations of the lipid matrix into highly curved intermediate structures. Bending stiffness of the lipid layers, progressively high with curvature, makes creation of such structures the major task for specialized protein machinery that has been evolved to perform membrane transformations at physiologically relevant length and time scales. Using dynamin 1, the GTPase implicated in membrane scission during endocytosis, as a prototype example we discuss the fission mechanisms in terms of enzymatic catalysis. We consider transient proteo-lipid complexes containing highly curved lipid structures as catalytic intermediates of the fission reaction. We distinguish two major intermediate structures, the pore and the hemi-fission connection, and describe how membrane binding, selfassembly and GTPase activity of dynamin 1 are coupled to creation and interconversion of these intermediates. We further identify major structural motifs making dynamin 1 the fission specialist and expand to general principles behind functional design of membrane fission catalysts.
Platform: Molecular and Cellular Neuroscience 783-Plat Modulation of Synaptic Vesicles Clustering by Axonal Tension Anthony Fan, Alireza Tofangchi, Taher Saif. University of Illinois at Urbana-Champaign, Urbana, IL, USA. Synaptic vesicles cluster at the presynaptic terminal to facilitate neurotransmitter release when an action potential arrives. It has been suggested—by severing (loss of tension) and subsequently pulling the axon (recovery of
Monday, February 13, 2017
Symposium: Biophysics of lncRNA 777-Symp Dynamic Temporal Control of Signaling Activated Gene Regulation Gregor Neuert. Vanderbilt University, Nashville, TN, USA. Many signal transduction and gene regulatory pathways are highly dynamic resulting in temporally varying activity of signaling proteins and time varying gene expression profiles. These time varying signaling profiles are important as these can direct different cellular phenotypes. Currently, our biophysical understanding of these temporal signaling profiles is very rudimentary and relies on the genetic manipulation of specific proteins or drug treatment regimes. Although protein numbers are often manipulated over a wide dynamic range using an inducible promoter, the activity of these signaling proteins may only be manipulated by mutations or drugs. One drawback of this approach is that genetic protein modification may result in a significant interference with the function of the cell, which may cause lethal phenotypes. Another drawback is that drugs are often not available for proteins of interest or have considerable off-target effect. In order to avoid these limitations, we developed a time varying perturbation approach that utilizes time varying ligand profiles to investigate the temporal properties of signaling and gene regulatory pathways, without genetic manipulation or drug treatments. To demonstrate the feasibility of this approach, we chose to interrogate an evolutionary conserved stress response pathway in yeast S. Cerevisiae, which enables to manipulate the intensity, duration and temporal activity of the signal transduction pathway in single cells. We are able to quantify signal transduction activation, signal transduction saturation and gene expression activation thresholds at high precision and demonstrate that the signaling dynamics is proportional to the first time derivative of the external perturbation profile. Because this approach is independent of the biological pathway or organism, it presents a general methodology to interrogate and control signaling and gene expression pathways non-invasively without the need for genetic or drug perturbations. 778-Symp The Ground-State of Promoter Directionality Revealed by a Functional Evolutionary Approach and Deep Learning Modeling Stirling Churchman. Department of Genetics, Harvard Medical School, Boston, MA, USA. Although the RNA polymerase (Pol) II machinery inherently initiates transcription in one direction, promoter regions are often ‘‘bidirectional’’ in vivo, giving rise to divergent RNA transcripts, many of which are noncoding and highly unstable. Here, we use a functional evolutionary approach to address whether bidirectional promoter regions are a mechanistic consequence of Pol II transcription or serve an evolved biological function. This involves nascent transcript mapping in S. cerevisiae strains containing large segments of foreign, and hence evolutionarily irrelevant, yeast DNA. A deep learning based model allows the unbiased identification of transcription start sites that emerge in the foreign DNA. Promoter regions in foreign species environments lose the directionality they have in their native species, indicating that DNA sequences and proteins co-evolve to promote directional transcription. Furthermore, fortuitous promoters emerge frequently in foreign DNA, and these produce equal transcription in both directions. Thus, without evolutionary pressure, the transcriptional ground state of promoter regions is intrinsically bidirectional. These results indicate that promoter regions are intrinsically bidirectional and are shaped by evolution to bias transcription of coding transcripts while suppressing non-coding transcriptional noise. 779-Symp How a lncRNA Shapes Chromatin Structure to Control Gene Expression Mitchell Guttman. Caltech, Pasadena, CA, USA. Mammalian genomes encode many thousands of long non-coding RNAs (lncRNAs) that play important roles in diverse biological processes. As a class, lncRNAs are generally enriched in the nucleus and specifically within the chromatin-associated fraction. Here, we will discuss the evidence that many nuclear-retained lncRNAs can interact with various chromatin regulatory proteins and recruit them to specific sites on DNA to regulate gene expression and shape three-dimensional nuclear organization. Specifically, we will discuss emerging mechanistic insights derived from the Xist lncRNA, a paradigm for lncRNA-mediated gene regulation, into how lncRNAs regulate gene expression by localizing to genomic target sites, recruiting regulatory complexes, and reshaping nuclear organization.
780-Symp Structure vs. Function: A Quantitative Analysis of Chromosome Architecture Luca Giorgetti. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. In animals, transcriptional regulation by distal genetic elements such as enhancers occurs in the context of the complex three-dimensional architecture of chromosomes. Experiments based on chromosome conformation capture such as Hi-C have shown that mammalian chromosomes are folded into a rich hierarchy of structural layers. At the core of this hierarchy, topologically associating domains (TADs) and their substructures appear to constrain and fine-tune the three-dimensional interactions of regulatory sequences, thus contributing to the establishment and maintenance of the correct gene expression patterns. We use a combination of quantitative experiments, genomic engineering and physical modeling to dissect the biophysical mechanisms by which chromosome structure and dynamics control transcriptional regulation.
Symposium: Catalyzed Membrane Fusion and Fission 781-Symp Know when to Hold ‘Em, Know when to Fold ‘Em; SM Proteins as Templates for SNARE Assembly Frederick Hughson. Department of Molecular Biology, Princeton University, Princeton, NJ, USA. A major focus of our research is the machinery that guides the fusion of vesicles during intracellular transport. These vesicles deliver their cargo via membrane fusion reactions executed by membrane-bridging SNARE complexes. The formation of SNARE complexes generally requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. Consequently, we hypothesize that SNARE complex assembly reactions in the cell are orchestrated by a set of ‘topologically aware’ chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing fourhelix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with - and sometimes as integral subunits of - the MTCs. Therefore, the goal of our work is to achieve an improved structural and mechanistic understanding of MTC and SM function in the assembly of fusogenic SNARE complexes. My presentation will focus on the SM proteins, and on our X-ray crystallographic and biochemical efforts to understand how they catalyze four-helix bundle formation. 782-Symp Catalytic Intermediates of Membrane Fission Vadim A. Frolov1, Pavel V. Bashkirov2, Anna V. Shnyrova1. 1 Biofisica Institute, Univ. Basque Country, Leioa, Spain, 2Federal Research and Clinical Centre of Physical-Chemical Medicine, Moscow, Russian Federation. Fusion and fission of cellular membranes entail localized transformations of the lipid matrix into highly curved intermediate structures. Bending stiffness of the lipid layers, progressively high with curvature, makes creation of such structures the major task for specialized protein machinery that has been evolved to perform membrane transformations at physiologically relevant length and time scales. Using dynamin 1, the GTPase implicated in membrane scission during endocytosis, as a prototype example we discuss the fission mechanisms in terms of enzymatic catalysis. We consider transient proteo-lipid complexes containing highly curved lipid structures as catalytic intermediates of the fission reaction. We distinguish two major intermediate structures, the pore and the hemi-fission connection, and describe how membrane binding, selfassembly and GTPase activity of dynamin 1 are coupled to creation and interconversion of these intermediates. We further identify major structural motifs making dynamin 1 the fission specialist and expand to general principles behind functional design of membrane fission catalysts.
Platform: Molecular and Cellular Neuroscience 783-Plat Modulation of Synaptic Vesicles Clustering by Axonal Tension Anthony Fan, Alireza Tofangchi, Taher Saif. University of Illinois at Urbana-Champaign, Urbana, IL, USA. Synaptic vesicles cluster at the presynaptic terminal to facilitate neurotransmitter release when an action potential arrives. It has been suggested—by severing (loss of tension) and subsequently pulling the axon (recovery of