Research
The cytoplasm is a mysterious jelly-like substance that sustains the biochemical reactions that are essential for life. How the cytoplasm organizes itself is one of the big remaining questions in biology. We use cell biological, biochemical, biophysical and genetic approaches and diverse model systems, such as yeast, Dictyostelium, and mammalian cells, to elucidate the molecular principles underlying the organization of the cytoplasm. We are particularly interested in understanding how the cytoplasm reorganizes itself upon environmental perturbations and stress. Stressed cells undergo changes on many levels to alter their physiology and metabolism; we are beginning to understand that many of these changes result from alterations in the structure and organization of the cytoplasm.
Research focus of the Alberti lab. The figure shows an idealized cell that transitions into a different physiological state upon stress. This transition is associated with changes in the organization of the cytoplasm and the formation of liquid- or solid-like condensates (gels, glasses, or crystals). In unstressed cells, the molecules are not interacting with each other and can thus be considered to be in a gas-like state (Unassembled). However, upon stress, they assemble into a condensed state (Assembled). Thus, the overall process has hallmarks of a phase transition.
Our recent work shows that stressed cells form many membraneless compartments in the cytoplasm via a biophysical process known as phase separation. However, the initially beneficial ability to form compartments becomes detrimental with increasing age, because compartment-forming have a tendency to misfold and aggregate and thus are closely tied to aging and the pathogenesis associated with age-related diseases such as amyotrophic lateral sclerosis (ALS). Thus, recent efforts in the lab are focused on understanding the molecular links between membraneless compartments and age-related diseases.
PROJECTS
Project 1: The chemistry and physics of the cytoplasm under stress
How do cells adapt to stress? We showed that the cytoplasm of stressed cells undergoes a phase transition from a liquid to a solid-like state (Munder et al., 2016). This phase transition is triggered by changes in physicochemical conditions such as fluctuations in cytosolic pH or temperature, which promote assembly of specific proteins and RNAs into cytoplasmic condensates.
What is the function of these condensates? We recently demonstrated that the yeast polyU-binding protein (Pub1) forms stress-protective condensates upon starvation or heat stress and that this is associated with cell cycle arrest (Kroschwald et al., 2018). Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). The different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates are reversible gels, whereas heat-induced condensates are solid-like and require chaperones for disassembly. Thus, different physiological stresses induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery.
In another recent study, we uncovered an unexpected function of the N-terminal prion domain of the canonical yeast prion protein and translation termination factor Sup35 (Franzmann et al., 2018). In stressed yeast, the Sup35 prion domain forms protective gels via pH-regulated phase separation followed by gelation (Figure 1). Gelation promotes cell survival by rescuing the essential translation factor from stress-induced damage. We propose that prion-like domains are protein-specific modifiers with chaperone-like functions that regulate protein phase behavior and protect proteins from damage.
Figure 1. Phase separation and gelation protects the essential translation termination factor Sup35 from stress-induced damage. (A) Sup35-GFP purified from insect cells forms liquid condensates in a pH-dependent manner. (B) Gel-like condensates of Sup35 as seen by Cryo-EM tomography. (C) Schematic describing the role of the N-terminal gel-forming domain (NM) of Sup35 during stress adaptation. In the presence of the gel-forming domain (left), Sup35 forms reversible gel condensates that protect the translation termination factor from damage. In the absence of the gel-forming domain (right), Sup35 forms irreversible aggregates that cause cell cycle arrest.
In the future, we aim to further characterize adaptive liquid-to-solid phase transitions of the cytoplasm. To this end, we will take a multi-scale approach, investigating these phase transitions on the level of entire cells, the cytoplasm and the level of macromolecules. We further aim to determine the function of various condensates that form when the cytoplasm solidifies. We hypothesize that condensate formation regulates the activity of proteins and protects proteins from damage, thus allowing stressed cells to adapt to the new environmental conditions, survive and recover from stress. We predict that this stress-protective system has similar significance as the well-established stress-protective system of molecular chaperones. More generally, we envisage that phase transitions are used across the kingdom of life to promote adaption to unfavorable environments. Future studies will therefore focus on the evolutionary forces that shape adaptive phase transitions in diverse organisms.
M. C. Munder, D. Midtvedt, T. Franzmann, E. Nüske, O. Otto, M. Herbig, E. Ulbricht, P. Müller, A. Taubenberger, S. Maharana, L. Malinovska, D. Richter, J. Guck, V. Zaburdaev, S. Alberti (2016). A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy. eLife, e09347, (2016).
S. Kroschwald, M. C. Munder, S. Maharana, T. M. Franzmann, D. Richter, M. Ruer, A. A. Hyman, S. Alberti. Different material states of Pub1 condensates define distinct modes of stress adaptation and recovery. Cell Reports, 23, 3327-3339, (2018).
T. M. Franzmann, M. Jahnel, A. Pozniakovsky, J. Mahamid, A. S. Holehouse, E. Nüske, D. Richter, W. Baumeister, S. W. Grill, R. V. Pappu, A. A. Hyman, S. Alberti. Phase separation of a yeast prion protein promotes cellular fitness. Science, 359, eaao5654, (2018).
Project 2: Membraneless compartments and disease-associated phase transitions
Proteins containing intrinsically disordered domains of low sequence complexity (also known as prion-like proteins) are frequently found in ribonucleoprotein (RNP) granules. What are the molecular properties of these proteins and what is their function? We demonstrated that the ALS-associated prion-like protein FUS forms dynamic RNP granules by liquid-liquid phase separation (Patel et al., 2015). We further found that liquid condensates assembled from patient-derived FUS show biophysical abnormalities and transition into an aberrant solid-like state. These findings suggest a molecular explanation for why these proteins are frequently associated with age-related diseases.
In another recent study, we used extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces for phase separation of FUS and related proteins (Wang et al., 2018). We found that phase separation of these proteins is driven primarily by interactions amongst tyrosine residues in prion-like domains and arginine residues in RNA binding domains. This work opens the door to predicting phase separation properties based on primary amino acid sequence.
How is the phase behavior of FUS and related prion-like proteins regulated in cells? FUS is largely soluble in the nucleus but it forms solid pathological aggregates when mislocalized to the cytoplasm. We found that this is due to the regulation of the solubility of FUS by RNA (Maharana et al., 2018). Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro (Figure 2). Reduction of nuclear RNA levels causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep FUS soluble. Furthermore, changes in RNA levels or RNA-binding abilities of FUS cause disease-causing aberrant phase transitions.
Figure 2.: RNA regulates the phase behaviour of the prion-like RNA-binding proteins FUS.FUS is in a diffuse and well-mixed state in the presence of high RNA concentrations but forms liquid condensates at intermediate RNA/protein ratios. Low RNA concentrations promote the conversion of FUS into a solid-like aggregated state.
In the future, we will investigate the phase behaviour of various disease-associated proteins. One important goal will be to dissect the molecular grammar of these proteins. This will allow us to determine how changes in the saturation concentration or material properties affect the biological function of condensates. In this context, we aim to understand on a deeper level how RNA regulates the phase behaviour of phase-separating proteins, focussing on features such as RNA multivalence and RNA secondary structure. Finally, we will investigate the role of ATP-driven machines such as molecular chaperones and helicases in regulating the material properties of condensates. By doing so, we hope to gain important insight into pathways that could delay the onset of age-related diseases.
A. Patel, H. K. Lee, L. Jawerth, S. Maharana, M. Jahnel, M. Y. Hein, S. Stoynov, J. Mahamid, S. Saha, T. Franzmann, A. Pozniakovski, I. Poser, N. Maghelli, L. Royer, M. Weigert, E. W. Myers, S. W. Grill, D. N. Drechsel, A. Hyman, S. Alberti. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell, 162, 1066-1077, (2015).
J. Wang, J. Choi, A. S. Holehouse, X. Zhang, M. Jahnel, S. Maharana, R. Lemaitre, A. Pozniakovski, 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, 174, 688-699 (2018).
S. Maharana, J. Wang, D.K. Papadopoulos, D. Richter, A. Pozniakovsky, I. Poser, M. Bickle, S. Rizk, M. Jahnel, Y. T. Chang, P. Tomancak, A. A. Hyman, S. Alberti. RNA buffers the phase separation behavior of prion-like RNA-binding proteins. Science, eaar7366, (2018).