OptoDroplets: Organelle formation controlled by Teleopto LED arrays

What are biomolecular condensates?

Biomolecular condensates are a unique class of organelles: they have no membranes. They can form, merge, split, and disappear in minutes, temporarily creating local incubators and assembly lines with properties very different from the bulk of the cells surrounding them. The local high concentrations of proteins and polynucleotides inside these condensates can both speed up and interfere with reactions, challenging the researchers trying to understand the rules of cell biology.  

Some condensates, such as the nucleolus and Cajal bodies, were first observed over a century ago, but others, such as processing bodies, PML bodies, and paraspeckles, were only discovered recently. It is only within the past few years that researchers have begun to understand that these organelles all share a common organizing principle: protein association drives the formation of gels which coalesce into the organelles themselves, which then behave according to the classic rules of phase separation and phase transition. These organelles condense in much the same way water vapor condenses into droplets on a window.

Why study biomolecular condensates?

This new understanding has led to condensates becoming a target for drug design. Dewpoint Therapeutics launched earlier this year, based on studies of stress granules. They seek to prevent temporary condensates of FUS protein from congealing into permanent aggregates, a driving force in amyotrophic lateral sclerosis (ALS). 

Liquid-liquid phase separation also has a role in gene expression: transcription factors have been found to rely on segregation inside condensates to initiate and control RNA production, yielding new targets for cancer therapies. The kinetics of ribosomal RNA processing is also proving to be dependent on the extent of gelation of the nucleolus.

Teleopto LED arrays and Biomolecular condensates

Clifford Brangwynne, Macarthur Fellow and Assoc. Prof. at Princeton University uses light to control the formation of condensates. Once activated by light, proteins like Cry2olig¹ oligomerize within seconds. By fusing Cry2olig to an RNA binding protein that drives condensation in the nucleus (NPM1), the BW lab created optoDroplets: light activated condensates held together by a meshwork of protein and nucleic acids.

Blue light from a Teleopto LEDA array causes these CRY2 fusions (opto-NPM1) to coalesce into a meshwork of proteins capable of turning the nucleolus of a cell into a tightly linked gel². The lab tunes the properties of the optoDroplets by adjusting the brightness: more light leads to more self-association and smaller pores in the meshwork. As the pores shrink, small proteins can still move through the hydrogel but larger molecules and complexes become trapped. This model allows the Brangwynne lab to study the effect of viscoelasticity on the formation of ribosomes and the processing of rRNA with just the press of a button. In a recent PNAS paper, they found that increasing the gelation of the nucleolus leads to the accumulation of larger rRNA precursors, while smaller precursors are depleted.

After the light is turned off, the condensates typically degenerate within 5 minutes. Fixing the cells while they are still illuminated allows the optoDroplets to be imaged and studied later, as shown in the figure below:

Postdoc Jorine Eeftens said that the Brangwynne lab used to use microscope mounted lasers to make condensates, but that only let them focus on a few cells at a time. The LEDA array allows them to activate many cells at once, greatly improving throughput in the lab. “We use it routinely, every day. We love working with it, the [LEDA] array allows us to use lots of cells, and then fix them for study. It’s our high throughput system.”

Biomolecular Condensates and Teleopto at the Woods Hole Physiology Course

The Woods Hole Marine Biology Laboratory discovery courses are intense, full-immersion summer courses for graduate students and postdocs. Students brainstorm, design and carry out their own projects – which frequently lead to publications. Ten years ago during a course led by Anthony Hyman and Brangwynne, then a postdoc in the Hyman lab, a project showed that P-granules behave like oil droplets when shearing forces are applied. The initial result from the Woods Hole class was followed up by Hyman and Brangwynne at Max Planck Institute, leading to a publication for both the students and the instructors. The paper shows that p-granule behavior follows the classic rules of phase separation and hinted at how this process could be involved in many more aspects of cellular behavior than previously thought³.

(1) Taslimi, A., Vrana, J. D., Chen, D., Borinskaya, S., Mayer, B. J., Kennedy, M. J., & Tucker, C. L. (2014). An optimized optogenetic clustering tool for probing protein interaction and function. Nature communications, 5, 4925.

https://doi.org/10.1038/ncomms5925

(2) Zhu, L., Richardson, T. M., Wacheul, L., Wei, M. T., Feric, M., Whitney, G., … & Brangwynne, C. P. (2019). Controlling the material properties and rRNA processing function of the nucleolus using light. Proceedings of the National Academy of Sciences, 116(35), 17330-17335.

https://www.pnas.org/content/116/35/17330 

(3)Brangwynne, C. P., Eckmann, C. R., Courson, D. S., Rybarska, A., Hoege, C., Gharakhani, J., … & Hyman, A. A. (2009). Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science, 324(5935), 1729-1732.

https://doi.org/10.1126/science.1172046