We investigate a fundamental aspect of intracellular organization – compartmentalization independent of lipid membranes. Canonical cellular compartments are confined within lipid membranes, but recent discoveries suggest that a plethora of cellular compartments form independent of membrane boundaries by liquid-liquid phase separation. We aim to understand how these ‘liquid-like’ compartments assemble and carry out distinct cellular functions. Currently we are investigating how the non-membrane-bound ‘nuage’ organelle, conserved in the germline of sexually reproducing animals, contributes to germ cell fate and fertility. Research in our group is multi-disciplinary – combines biochemistry, biophysics, structural biology, in vitro reconstitution, and in vivo studies in C. elegans.
Cells contain over twenty non-membrane-bound compartments which must carry out unique biochemical functions to maintain cellular homeostasis. Lipid membranes limit free exchange of macromolecules between canonical cellular compartments and their surroundings allowing specialized biochemical environments to develop within these compartments. It is therefore puzzling how non-membrane-bound compartments carry out unique biochemistry in spite of the constant exchange of macromolecules with their surroundings. Clues into this puzzle are only beginning to emerge.
Many non-membrane-bound compartments have recently been proposed to assemble by a process called liquid-liquid phase separation. This process is analogous to the separation of oil and vinegar phases in vinaigrette. Under favorable conditions, certain proteins and RNAs phase separate from the surrounding cytoplasm or nucleoplasm to assemble intracellular ‘liquid-like’ compartments.
Our lab played a significant role in the discovery of the molecular mechanisms of assembly of cellular ‘liquid-like’ compartments and how such assembly can be spatially regulated (Alberti*, Saha* et al., 2018; Saha et al., 2016). In vitro reconstitution approaches have shown that among the hundreds of proteins and RNAs that reside in these compartments, phase separation of only one or a few “scaffold” protein or RNA drive compartment assembly. Other proteins and RNAs partition into the scaffold phase as “clients” to modulate biological function.
Much remains to be learnt about liquid-like intracellular compartments. For instance, what principles define their precise macromolecular composition and material properties? How is this appropriate for their biological function? Interestingly, some proteins and RNAs can partition simultaneously into two or more phase separated compartments that differ in their overall composition. How can these compositionally distinct compartments that share components coexist within the common cytoplasm or nucleoplasm?
A canonical liquid-like compartment is the ‘Nuage’, present in the germline of sexually reproducing organisms (including humans). The germline ensures continuance of animal species by making the totipotent embryo for the next generation. Nuage is present specifically in the germline and absent from somatic cells. Mutations in nuage proteins are known to generate teratomas and cause infertility. In spite of the discovery of germ granule/Nuage over a century ago, a concrete definition of its function at the molecular mechanistic level is still lacking. Nuage has been implicated in genome surveillance via the piRNA pathway and more recently in RNA-directed transgenerational epigenetic inheritance – mainly based on the observation that some proteins and RNAs involved in these processes are present in nuage. Why it is important to have these proteins/RNAs in a phase-separated state in nuage remains unclear.
A major bottleneck towards addressing molecular mechanisms of Nuage function has been the inability to isolate them from in vivo sources. Dilutions that generally accompany purification strategies led to dissolution of liquid-like Nuage. We have shown that P granule (Nuage)-like phases can be reconstituted in vitro from purified components – thereby providing a way around the long-standing bottleneck (Saha et al., 2016). Extensive investigation from the perspective of biochemistry is therefore now possible to understand how Nuage interacts with its surroundings, how the internal environment within Nuage influences the activity of its components, and how this altogether contributes to normal germline development and totipotency.
Our research will provide significant insights into two of the central questions in phase separation-dependent intracellular organization – 1) Rules that determine precise macromolecular composition i.e. specificity, and 2) Importance of phase separation for biological function. Phase separation has now been implicated in a wide range of fundamental biological processes and diseases - cellular DNA-damage response, RNA processing including transcription, splicing, translation and storage, biology of non-coding RNAs, signal transduction, T-cell activation, viral infection, neurodegeneration, and cancer. Therefore, the general principles about phase separation uncovered in our research will help with understanding the mechanisms of a wide range of vital biological processes.
Findings from our research could contribute to new cellular reprogramming strategies relevant to regenerative medicine. State-of-the-art techniques on reprograming somatic cells into stem cell-like states, or direct reprograming of somatic cells into differentiated cells of other somatic lineages are often very inefficient. There is a need for new tools that could be used in combination with existing approaches for better control and efficiency in cellular reprograming. Development of new tools depend on discovery of novel mechanisms of cell fate decisions. Studying Nuage in germ cells is interesting in this respect, since this organelle helps maintain germ cell fate through yet to be discovered mechanisms. Therefore, our research studying the foundations of a germ cell-specific phenomenon may lay the groundwork for understanding problems with fertility as well as provide new insights into how to better reprogram cells into stem cell-like states.
Stela Jelenic, Janos Bindics, Philipp Czermak, Balashankar R Pillai, Martine Ruer, Alex S Holehouse, Shambaditya Saha (2022). Composition can buffer protein dynamics within liquid-like condensates BioRxiv.
Alberti, S., Saha, S., Woodruff, JB., Franzmann, TM., Wang, J., Hyman, AA. (2018). A User's Guide for Phase Separation Assays with Purified Proteins. J Mol Biol. 430(23):4806-4820
Saha, S., Weber, CA., Nousch, M., Adame-Arana, O., Hoege, C., Hein, MY., Osborne-Nishimura, E., Mahamid, J., Jahnel, M., Jawerth, L., Pozniakovski, A., Eckmann, CR., Jülicher, F., Hyman, AA. (2016). Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism. Cell. 166(6):1572-1584.e16