Check out our new user’s guide for phase separation assays with purified proteins!

Highlights

  • Membrane-less organelles form by phase separation
  • Membrane-less organelles can be reconstituted from minimal components
  • Phase-separating proteins are difficult to purify and handle
  • We provide guidelines and protocols for working with phase-separating proteins

 

The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high quality proteins, standardized procedures, and generally agreed upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.

reference: https://doi.org/10.1016/j.jmb.2018.06.038

 

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Jie’s paper on the molecular grammar of phase separation is out!

Our latest publication is out now in Cell! Congratulations to Jie and all the co-workers.

 

In this work, Jie Wang and colleagues worked with a family of prion like proteins (FUS family proteins) and suggest rules that define their phase separation behavior. In cells, FUS family proteins can phase separate into liquid like organelles. However, they can form as well aggregates that are linked to neurodegenerative diseases like ALS. Understanding and predicting their phase behavior will lead to a better understanding of their cellular functions.

 

For a scientific summary of his work and the original publication see below. Click here for a more general summary.

 

Fig. Hierarchical organization that gives rise to formation of condensates depicted here as spherical droplets

 

  • Phase separation of FUS requires both the N-terminal PLD and the C-terminal RBD
  • Tyrosine and arginine govern the saturation concentration of phase separation
  • Glycine maintains liquidity, whereas glutamine and serine promote hardening
  • An associative polymer model predicts the phase behavior of FUS family proteins

 

Fig. The number of arginines and tyrosines determine the phase behavior of FUS family proteins

 

Summary

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.

 

Original Publication

J. Wang, J.M. Choi, A.S. Holehouse, X. Zhang, M. Jahnel, R. Lemaitre, S. Maharana, A. Pozniakovsky, D. Drechsel, I. Poser, R.V. Pappu, S. Alberti, A.A. Hyman: A molecular grammar underlying the driving forces for phase separation of prion-like RNA binding proteins. Cell, In Press (2018)

https://doi.org/10.1016/j.cell.2018.06.006

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Buffering disease: How the nucleus keeps proteins in check

High RNA concentration maintains RNA-binding proteins in solution and prevents pathological aggregates

 

 

 

Many age-related diseases affect the nervous system. One prominent example of a neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS). The brain tissue of ALS patients typically shows aggregates of so-called prion-like RNA-binding proteins. In the nucleus, these RNA-binding proteins are floating in solution, but when they are located outside of the nucleus, in the cytoplasm, they often form solid pathological aggregates. Researchers from the lab of Simon Alberti and Tony Hyman at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden wanted to investigate why these proteins never aggregate in the nucleus and what keeps them soluble there? Uncovering this mechanism may help to dissolve pathological aggregates in the cytoplasm…read more

 

Shovamayee Maharana, Jie Wang, Dimitrios K. Papadopoulos, Doris Richter, Andrey Pozniakovsky, Ina Poser, Marc Bickle, Sandra Rizk, Jordina Guillén-Boixet, Titus Franzmann, Marcus Jahnel, Lara Marrone, Young-Tae Chang, Jared Sterneckert, Pavel Tomancak, Anthony A. Hyman, Simon Alberti: RNA buffers the phase separation behavior of prion-like RNA binding proteins, Science, 12 Apr 2018

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Richard’s review on controlling non-membrane-bound organelles is out

Richard stayed in the lab just about a year but his contributions to the Science of the Hyman lab, not forgetting his many social contributions, have been quite big! Now he wrote a review with Tony for the Philosophical Transactions B on controlling non-membrane-bound organelles. They discuss energetically favourable interactions that could drive condensation, and on the basis of that, make qualitative predictions about how cells may control compartmentalization by condensates.

Controlling compartmentalization by non-membrane-bound organelles. Wheeler RJ, Hyman AA. Philos Trans R Soc Lond B Biol Sci. 2018 May 26;373(1747). pii: 20170193. doi: 10.1098/rstb.2017.0193. Review. PMID:29632271

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Edgar is giving a talk at the CSHL meeting Protein Homeostasis in Health and Disease

The Cold Spring Harbor meeting on Protein Homeostasis is taking place from April 17th-21th and Edgar will speak about how Molecular chaperones control the physical state of membrane-less compartments. Go Edgar!

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Congratulations to Stephanie for her successful collaboration with the Zerial lab!

Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria.

Hsu F, Spannl S, Ferguson C, Hyman AA, Parton RG, Zerial M.

Elife. 2018 Feb 22;7. pii: e32282. doi: 10.7554/eLife.32282

 

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS.

 

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Congratulations to Sina and Adam for their fellowships!

Sina and Adam received both each an EMBO postdoc fellowship as well as a Marie Curie postdoc fellowship. This is a great success for both of them but as well for the lab. And yes, of course we celebrated it…

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ASAPbio cenference: Transparency, Recognition, and Innovation in Peer Review in the Life Sciences

Tony will be part of the ASAPbio conference on Transparency, Recognition and Innovation in Peer Review in the Life Sciences taking place February 7th to 9th at the HHMI headquarters in Chevy Chase, Maryland. Several aspects of peer review will be discussed in the meeting and importantly, you can follow the conference by live video streaming.

Check out two essays Tony wrote on Peer Feedback and the defence of the Peer review

 

ASAPbio (Accelerating Science and Publication in biology) is a scientist-driven initiative to promote the productive use of preprints in the life sciences.

Board of Directors
Ron Vale (President)
Cynthia Wolberger (Vice President)
James Fraser (Secretary & Treasurer)
Prachee Avasthi
Daniel Colón-Ramos
Tony Hyman
Heather Joseph
Harlan Krumholz
Maria Leptin
Harold Varmus
Dick Wilder (non-voting)

Director
Jessica Polka

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Congratulations to Titus and coworkers for his Science paper

Prions are self-propagating protein aggregates that can be transmitted between cells. The aggregates are associated with human diseases. Indeed, pathological prions cause mad cow disease and in humans Creutzfeldt-Jakob disease. The aggregation of prion-like proteins is also associated with neurodegeneration as in ALS. The regions within prion-like proteins that are responsible for their aggregation were termed prion-like domains. Despite the important role of prion-like domains in human diseases, a physiological function has remained enigmatic…read more

 

Cryo-Electron Microscopy Image of a biomolecular condensate of a Prion Protein

 

 

Reference:

Titus M. Franzmann, Marcus Jahnel, Andrei Pozniakovsky, Julia Mahamid, Alex S. Holehouse, Elisabeth Nüske, Doris Richter, Wolfgang Baumeister, Stephan W. Grill, Rohit V. Pappu, Anthony A. Hyman und Simon Alberti, Phase separation of a yeast prion protein promotes cellular fitness, Science, (359) 5 January 2018.

DOI: 10.1126/science.aao5654

 

 

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Parkinson Broschüre ist jetzt erhältlich

Ein Forscherteam unseres Hauses hat eine bahnbrechende Entdeckung gemacht, nämlich dass die Gen-Produkte D-Laktat und Glykolat positive Effekte auf angegriffene Zellen haben. Genau das ist das Problem bei der Krankheit Parkinson: Nervenzellen sterben ab, wenn ihre Kraftwerke, die Mitochondrien, nicht mehr richtig funktionieren…mehr

soon this brochure will be available in English as well.

 

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