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.

go to the pdf

Posted in Uncategorized | Comments closed

Phase separation buffers noise in cells



We are excited to share our pre-print which explores how phase separation affects noise in cells. The question of how cells manage to tightly control protein concentrations has been a long-standing challenge in biology. In collaboration with the Zechner and Jülicher groups, we now show in theory and experiments that liquid-liquid phase separation (LLPS) can be a very effective mechanism to buffer protein concentrations against gene expression noise.


The protein condensates used in our study dissolve during mitosis so that the protein concentration in the dilute, bulk phase is no longer buffered and variability becomes apparent.

In conclusion our results suggest a novel role of phase separation for gene regulation and biological information processing.

See below a mitosis movie of the tagged DDX4 protein construct


Click for the pdf of the preprint or go to the biorxiv page

Posted in Uncategorized | Comments closed

Rheology of protein condensates using optical traps

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquidlike phases. These protein condensates serve as simple models for the investigation of the more complex membraneless organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important.


Fig: scheme of the set up based on a dual optical trap

*

Louise and her coworkers now developed a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of P-granule protein PGL-3 condensates as a function of salt concentration. Have a look at the pdf or follow the link below.

*

 

Posted in Uncategorized | Comments closed

How do you choose a research topic?

Choosing a research topic is an important decision at any level. The choice shapes decisions about what graduate lab to join, which post-doctoral position to pursue, how to start an independent lab, and what companies might make good employers. The thing to remember is that “the choice” can, and perhaps should, be made many times during a career, and each time, it can take an exciting new turn. Lara Szewczak sat down with Amy Gladfelter and Tony Hyman to talk about what it takes for a researcher to pivot—to decide that they want to embark on a new line of research. What does it mean at different points in a career, and how can you motivate colleagues to follow?

 

that’s where the pivot begins—is the mystery. I think the pace and the pressure to produce—the sense that you need to be in the thing that is getting published right now—is covering up a lot of the mystery.You need mentors to say, ‘No, this is how you will actually get grants. This is how you’ll get recognized by something a bit different.’

 

Read the interview of Amy Gladfelter and Tony!

Posted in Uncategorized | Comments closed

Phase Transitions form Virus Replication Compartments in Cells

Zoltan’s paper is out!

Former postdoc Zoltan worked with the Whelan lab on VSV (Vesicular Stomatitis Virus). RNA viruses like VSV compartmentalize their replication machinery to evade detection by the host. However, it has been unclear how the virus can concentrate the machinery for RNA synthesis. Now it is shown that the replication compartments have liquid like properties and form by phase separation. Liquid-liquid phase separation (LLPS) seems to play an important role in host-pathogen interactions.

Dynamics of VSV inclusions

 

Phase Transitions Drive the Formation of Vesicular Stomatitis Virus Replication Compartments. Heinrich BS, Maliga Z, Stein DA, Hyman AA, Whelan SPJ. MBio. 2018 Sep 4;9(5). pii: e02290-17. doi: 10.1128/mBio.02290-17.

 

 

Posted in Uncategorized | Comments closed

Former postdoc Richard opens his own lab in Oxford

Congratulations to Richard! Check out his website

Posted in Uncategorized | Comments closed

We are organizing an EMBO Practical Course for studying phase separation in biology!

The course takes place 05 – 13 February 2019 in Dresden at the MPI-CBG,

register until 1st Oct. 2018, we are looking forward to seeing you here!

 

#EMBOphaseseparation / facebook

 

About the Practical Course

The field of cell biology is in the midst of a revolution in the understanding of how cells are biochemically organized. In recent years, research has shown that liquid-liquid phase separation is a critical organizing principle for cells, improper regulation of which can lead to dysfunction and disease. There are many exciting open research questions in this field, and we aim to provide students with an essential toolkit for addressing them. This course will provide high quality instruction in the physical chemistry theories underlying biological phase separation, followed by hands on practical training in the assays and cutting-edge techniques used in this emerging field. Dresden is a hub for phase separation research, and in addition to our local experts in biophysics and cell biology, our speakers and instructors will also include global authorities on these topics. Following the course, participants should be able to apply their newly learned techniques to their own projects and research questions. We hope that this course will establish the best practices in the field of biological phase separation, which the students can spread more widely at their home and future institutions.

 

Posted in Uncategorized | Comments closed

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

Posted in Uncategorized | Comments closed

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

Posted in Uncategorized | Comments closed

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

Posted in Uncategorized | Comments closed
  • Selected Videos


    iBio video about P granule formation


    An interview with Tony about Phase Transitions and Disease


    Phase separation in cell polarity: Saha et al, Cell 2016


    Encouraging Innovation, iBiology.org


    The genetics linking temperature and fertility in worms: Leaver et al, Biology Open 2016


    Cell PaperFlick on Phase Transitions in Disease


    Check out this playlist to watch all the videos in our "Two Minute Talk" Series


    Ways of Growing, a film created for the MitoSys Project


    What is a Discovery?


    Embryonic Development of C.elegans

  • News Archive