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Phase Separation

Available via

WEBINAR

Phase Separation in Biology and Disease

Wednesday, February 20, 2019, 8:30 AM - 5:00 PM

The New York Academy of Sciences, 7 World Trade Center, 250 Greenwich St Fl 40, New York

Presented By

The Chemical Biology Discussion Group

The New York Academy of Sciences

 

Liquid-liquid phase separation of cytoplasmic and nuclear components into biomolecular condensates is emerging as a fundamental mechanism of cellular organization. These condensates are spatially segregated, but open systems, which can serve as specialized regions for biochemical reactions, dynamically sequester components or act as organizational hubs for signaling networks.

Phase separation regulates processes as diverse as transcription, stress granule formation and actin polymerization. Moreover, an accumulating body of evidence suggests that dysregulation of these phase transitions may contribute to neurodegenerative diseases, including ALS and prion diseases, as well as to cancer.

This one day symposium will bring together scientists from academia and industry to dissect the latest advances in the field of biological phase separation and discuss the implications for human disease.

Registration

Member
By 01/11/2019
$90
After 01/11/2019
$130
Nonmember Academia, Faculty, etc.
By 01/11/2019
$180
After 01/11/2019
$260
Nonmember Corporate, Other
By 01/11/2019
$250
After 01/11/2019
$350
Nonmember Not for Profit
By 01/11/2019
$180
After 01/11/2019
$260
Nonmember Student, Undergrad, Grad, Fellow
By 01/11/2019
$100
After 01/11/2019
$145
Member Student, Post-Doc, Fellow
By 01/11/2019
$50
After 01/11/2019
$70
Member
$30
Nonmember Academia, Faculty, etc.
$65
Nonmember Corporate, Other
$85
Nonmember Not for Profit
$65
Nonmember Student, Undergrad, Grad, Fellow
$45
Member Student, Post-Doc, Fellow
$15
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Scientific Organizing Committee

Clifford Brangwynne
Clifford Brangwynne, PhD,
Princeton University
Jason Imbriglio, PhD,  Merck
Jason Imbriglio, PhD,
Merck
Neal Zondlo, PhD,  University of Delaware
Neal Zondlo, PhD,
University of Delaware
Sara Donnelly, PhD, The New York Academy of Sciences
Sara Donnelly, PhD, The New York Academy of Sciences
Sonya Dougal, PhD, The New York Academy of Sciences
Sonya Dougal, PhD, The New York Academy of Sciences

Speakers

Clifford Brangwynne, PhD,  Princeton University
Clifford Brangwynne, PhD,
Princeton University
Zhijian "James" Chen, PhD,  University of Texas, Southwestern
Zhijian "James" Chen, PhD,
University of Texas, Southwestern
Geraldine Seydoux, PhD,  Johns Hopkins School of Medicine
Geraldine Seydoux, PhD,
Johns Hopkins School of Medicine
Abby Dernburg, PhD,  University of California, Berkeley
Abby Dernburg, PhD,
University of California, Berkeley
Rohit Pappu, PhD  Washington University in St. Louis
Rohit Pappu, PhD
Washington University in St. Louis
Tanja Mittag
Tanja Mittag, PhD

St. Jude Children's Research Hospital

Nicolas Fawzi, PhD,  Brown University
Nicolas Fawzi, PhD,
Brown University
Martin Jonikas, PhD,  Princeton University
Martin Jonikas, PhD,
Princeton University
David Cowburn, PhD, DSc,  Albert Einstein College of Medicine
David Cowburn, PhD, DSc,
Albert Einstein College of Medicine





Wednesday

February 20, 2019

8:30 AM

Continental Breakfast and Registration

9:00 AM

Introduction and Welcome Remarks

Speakers

Sara Donnelly, PhD
The New York Academy of Sciences
Neal Zondlo, PhD
University of Delaware

Session 1: Composition and Regulation of Biomolecular Condensates

Session Chairperson
Neal Zondlo, PhD, University of Delaware
9:15 AM

Lighting up Phase Transitions

Speaker

Clifford Brangwynne, PhD
Princeton University

In this talk I will discuss our work to understand and engineer intracellular phase transitions, which play an important role in organizing the contents of living cells. Membrane-less RNA and protein rich condensates are found throughout the cell, and regulate the flow of genetic information. We've shown that liquid-liquid phase separation (LLPS) underlies the assembly of these structures. LLPS driven by intrinsically disordered protein regions (IDRs) explains many condensate features, for example the internal subcompartments of the nucleolus, which has important consequences for sequential ribosomal RNA processing. Our lab has developed a suite of new approaches, which use light to enable spatiotemporal control of intracellular phase transitions, allowing us to engineer the assembly and disassembly of these structures within defined subregions of the cytoplasm and nucleus. We are now using these tools to quantitatively map intracellular phase diagrams for the first time, providing unprecedented access to the biophysical principles underlying RNP condensate self-assembly. This approach has also begun to yield rich insights into the link between intracellular liquids, gels, and the onset of pathological protein aggregation.

9:45 AM

Regulation of RNA Granule Dynamics by Intrinsically-disordered Proteins

Speaker

Geraldine Seydoux, PhD
Johns Hopkins School of Medicine

RNA granules are RNA-protein condensates that form in the cytoplasm or nucleoplasm of cells. We are studying the P granules of C. elegans as a model to uncover principles of RNA granule assembly and function. We have identified a small family of intrinsically-disordered proteins (MEGs) that drive P granule assembly in C. elegans embryos. I will present our recent findings on how the MEGs recruit mRNAs to P granules.

10:15 AM

Networking Coffee Break

10:45 AM

Characterization of Multivalent Interactions of Proteins

Speaker

David Cowburn, PhD, DSc
Albert Einstein College of Medicine

Intrinsically disordered proteins (IDPs) play important roles in many biological systems, including phase
seperated low complexity domain. Given the vast conformational space that IDPs can explore, the
thermodynamics of the interactions with their partners is closely linked to their biological functions.
Intrinsically disordered regions of Phe–Gly nucleoporins (FG Nups) that contain multiple
phenylalanine–glycine repeats are of particular interest, as their interactions with transport factors (TFs)
underlie the paradoxically rapid yet also highly selective transport ofmacromoleculesmediated bythe
nuclear pore complex. We used NMR and isothermal titration calorimetry to thermodynamically
characterize these multivalent interactions. These analyses revealed that a combination of low FG
motif affinity and the enthalpy–entropy balance prevents high avidity interaction between FG Nups and
TFs, whereas the large number of FG motifs promotes frequent FG–TF contacts, resulting in enhanced
selectivity. Our thermodynamic model underlines the importance of functional disorder of FG Nups. It
helps explain the rapid and selective translocation of TFs through the nuclear pore complex and further
expands our understanding of the mechanisms of “fuzzy” interactions involving IDPs.

11:15 AM

Lessons from the Phase Behavior of Block Co-polymeric Proteins with Low Complexity Domains

Speaker

Rohit Pappu, PhD
Washington University in St. Louis

There are numerous examples of  of multivalent proteins with intrinsically disordered low complexity domains working in tandem with folded domains to determine the driving forces for the phase separation. The exon1 spanning region of huntingtin (Httex1) encodes for a block co-polymeric sequence comprising of two ultra low complexity domains encompassing a polyglutamine (polyQ) and polyproline tracts, respectively. Work over the past few years has uncovered a complex, concentration and polyQ length dependent phase diagram that includes spheroidal micellar structures and rod-like fibrils. A detailed thermodynamic description and an emerging dynamical description are paving the way for uncovering the rules that underlie the interplay between aggregation and phase separation and the transitions between soluble, liquid-like assemblies and insoluble fibrillar deposits. These complexities of Httex1 phase diagrams are being brought to bear on understanding the onset and progression of Huntington's disease (HD). In addition, recent work has focused on the effects of ligand binding in modulating Httex1 phase behavior through a phenomenon known as polyphasic linkage. The findings uncovered for Httex1 and the impact of polyphasic linkage on its phase behavior are directly relevant to a range of protein systems with similar domain architectures. This talk will go over the specific findings for Httex1 that have been uncovered through a combination of theory, computation, and experiment and use these findings to highlight concepts that are likely to be relevant to describe and design the phase behaviors of other multivalent proteins.

Session 2: Data Blitz Talks

Session Chairperson
Neal Zondlo, PhD, University of Delaware
11:45 AM

Disease-linked Mutations Promote UBQLN2 Oligomerization and Phase Separation

Speaker

Carlos A. Castañeda, PhD
Syracuse University
11:55 AM

Deciphering the Chemical Drivers of Phase Separation and Gelation in FG- repeat Peptides

Speaker

Steven J. Metallo, PhD
Georgetown University
12:05 PM

Phosphorylation-Mediated Disassembly and Clearance of Amyloid-Like Assemblies in Meiosis

Speaker

Luke E. Berchowitz, PhD
Columbia University Irving School of Medicine
12:15 PM

Networking Lunch and Poster Session

Speakers

Odd Numbered Posters
12:45 PM - 1:15 PM
Even Numbered Posters
1:15 PM - 1:45 PM

Session 3: Regulation of Physiological Processes by Phase Separation

Session Chairperson
Clifford Brangwynne, PhD, Princeton University
1:45 PM

DNA-induced phase separation of cGAS

Speaker

Zhijian "James" Chen, PhD
University of Texas Southwestern
2:15 PM

Structure and Biogenesis of the Eukaryotic CO2-Concentrating Organelle, the Pyrenoid

Speaker

Martin Jonikas, PhD
Princeton University

Approximately one-third of global carbon-fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco, and enhances carbon-fixation by supplying Rubisco with a high concentration of CO2. The molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. To advance our understanding of the pyrenoid and of photosynthetic organisms more broadly, we have developed new tools for the unicellular model alga Chlamydomonas. These tools include the world’s first genome-wide collection of mapped mutants in any single-celled photosynthetic organism, as well as methods for high-throughput localization of proteins and identification of protein-protein interactions. By applying these tools, we increased the number of known pyrenoid components from 6 to over 80, and discovered the existence of three new protein layers in the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We discovered that an abundant pyrenoid protein, Essential Pyrenoid Component 1 (EPYC1), works as a molecular glue that binds Rubisco holoenzymes together to form the matrix at the core of the pyrenoid. Furthermore, contrary to longstanding belief that the pyrenoid matrix is a solid structure, we discovered that the matrix behaves as a liquid droplet, which mixes internally, divides by fission, and dissolves and condenses during the cell cycle. Our data provide insights into pyrenoid protein composition, structural organization and biogenesis. Working with our collaborators in the Combining Algal and Plant Photosynthesis project, we aim to transfer algal pyrenoid components into higher plants to enhance carbon fixation and yields in crops.

2:45 PM

Phase Separation Mediates Intracellular Pattern Formation

Speaker

Abby Dernberg, PhD
University of California Berkeley

The cell division process of meiosis partitions a diploid genome to produce haploid cells, and is essential for sexual reproduction. During meiotic prophase, each chromosome pairs with its homolog. Paired homologs then “zipper up” via assembly of a unique protein polymer, the synaptonemal complex (SC), along their interface. Through in vivo imaging in Caenorhabditis elegans, we discovered that the SC behaves as a liquid crystal, and have been investigating the implications of this finding.

A longstanding mystery has been the regulation of meiotic recombination: each pair of chromosomes must undergo at least one crossover, but the total number of crossovers is typically very low, often just one per pair. The SC plays a crucial role in crossover control. We have now found that ZHP-1–4, members of a widely conserved family of meiotic RING finger proteins, diffuse within the SC and interact with components anchored at recombination intermediates to pattern crossovers.

In 1952, the mathematician Alan Turing first proposed that biological patterns might arise through coupled feed-forward and feedback interactions within a diffusive medium. Such “reaction-diffusion” mechanisms can operate on any spatial scale. They are thought to underlie pigmentation patterns and the regular spacing of hair, feathers, and fish scales, and have also been proposed to mediate intracellular patterning and symmetry breaking. Liquid-liquid (or liquid crystalline) phase separation (LLPS) gives rise to subcellular compartments of varying geometries that concentrate biomolecules and impose boundaries to their diffusion. We propose that LLPS may create conditions that enable formation of diverse subcellular patterns.

Session 4: Phase Separation in Disease

Session Chairperson
Jason Imbriglio, PhD, Merck
3:15 PM

Networking Coffee Break

3:45 PM

Phase Separation for Proteostasis in Normal and Cancerous Cells

Speaker

Tanja Mittag, PhD
St. Jude Children's Research Hospital

Liquid–liquid phase separation is the biophysical driving force for the formation of membrane-less organelles in cells, such as stress granules, nucleoli and nuclear speckles. Current open questions are: (i) How is phase separation propensity encoded in the protein sequence, (ii) are dense liquid droplets used as reaction compartments in the cell, and (iii) is physiological phase separation disrupted in disease states? To address these, we study the tumor suppressor Speckle-type POZ protein (SPOP), a substrate adaptor of a ubiquitin ligase, which localizes to different liquid membrane-less organelles in the cell nucleus. In these organelles, SPOP encounters its substrates but its recruitment mechanism to the organelles is not understood. Here, we show that SPOP undergoes LLPS with substrate proteins, and that this mechanism underlies its recruitment to membrane-less organelles. Multivalency of SPOP and substrate for each other drive their ability to phase separate. We present evidence that the SPOP/substrate assemblies are active ubiquitination compartments in vitro and in cells. SPOP cancer mutations reduce the propensity for phase separation. We propose that SPOP has evolved a propensity for phase separation in order to target substrates localized in membrane-less compartments. Using phase separation for proteostasis may allow setting an upper limit for substrate protein levels in cells.

Coauthors: Jill J. Bouchard[1], Joel H. Otero[1], Daniel C. Scott[1], Elzbieta Szulc[2], Erik W. Martin[1], Nafiseh Sabri[1], Daniele Granata[3], Kresten Lindorff-Larsen[3], Xavier Salvatella[2,4], Brenda A. Schulman[5,6].

1. St. Jude Children’s Research Hospital, Memphis.
2. The Barcelona Institute of Science and Technology, Barcelona.
3. University of Copenhagen.
4. ICREA, Barcelona.
5. Max-Planck Institute of Biochemistry, Martinsried/Munich.
6. Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis.

4:15 PM

Functional and Pathological RNA-binding Protein Phase Separation with Atomic Detail

Speaker

Nicolas Fawzi, PhD
Brown University

Cells use various mechanisms to organize reactions and sequester proteins, RNA, and chromatin for transcription, processing, and localization. One emerging mechanism is liquid-liquid phase separation mediated by the association of the disordered domains of RNA binding proteins. RNA-binding proteins FUS, TDP-43, and hnRNPA2 are all associated with RNA granule assembly and all form inclusions in amyotrophic lateral sclerosis and multisystem proteinopathy, respectively. Using these proteins as models, we probe their molecular structure along the assembly pathway and the structural changes caused by disease mutations and post-translational modifications. Using nuclear magnetic resonance (NMR) spectroscopy and molecular simulation, we see their structure and interactions with atomic resolution. These findings are paired with microscopy and turbidity experiments and cell assays to assess the effect of posttranslational modifications and mutations on phase separation, aggregation, toxicity and splicing function. We find that low complexity domains remain predominantly unstructured both before and after phase separation. The exception is TDP-43 where phase separation and protein function is enhanced by a globular domain and an alphahelical region whose helical extent increases and extends upon phase separation. Arginine methylation and phosphorylation disrupts phase separation, aggregation, and cellular toxicity. Our work points to the potential for post-translational modification to alter assembly, function, and pathological interactions of disease-associated disordered domains.

4:45 PM

Closing Remarks

Speaker

Jason Imbriglio, PhD
Merck
4:50 PM

Networking Reception and Poster Session

5:15 PM

F1000 Poster Prize Presentation

Speaker

Clifford Brangwynne, PhD
Princeton University
5:50 PM

Adjourn

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