Amy Gladfelter: Encoding temperature sensitivity in biomolecular condensates

Live blogger: Sadie Gugel 

Editors: Varsha Shankar and Jennifer Baker

This piece was written live during the 7th annual RNA Symposium, “From Molecules to Medicines,” hosted by the University of Michigan’s Center for RNA Biomedicine. Follow MiSciWriters’ coverage of this event on Twitter with the hashtag #umichrna.

The nucleus, the endoplasmic reticulum, and the mitochondria are organelles likely familiar to many of us from biology class. These structures are separated from the rest of the cell by membranes and are used by eukaryotic cells to compartmentalize and organize molecules that support specific cell functions. While these organelles are certainly important, Dr. Amy Gladfelter and her group are interested in a different kind of cellular organization: biomolecular condensates. 

Condensates are sequestered groups of molecules, and in her talk today, Dr. Gladfelter focuses on those formed between RNA and protein. Specific interactions between these molecules drive the group of molecules to undergo phase separation and a condensate can form. In this process, condensates form droplet-like structures that no longer mix with the rest of the liquid in the cell, like oil droplets suspended in water. This allows for separation and localization of specific molecules in regions of the cells where they can execute their functions. In this way, condensates serve similar functions to organelles, without being bound by a membrane. 

Condensates are particularly critical for fungi that form syncytia – single cells with more than one nucleus. In a typical cell with one nucleus, the signals for regulating nuclear division (the process of a cell copying its genetic information and splitting the nucleus into two) can be spread all throughout the cell until they reach the nucleus and initiate this process in response to environmental conditions. However, syncytia are very large, which means nuclei in different regions of the cell may need to respond to different conditions at different times. So how do these cells regulate this process for different nuclei within the same cell? 

While studying fungal syncytia in her postdoctoral research, Dr. Gladfelter made an astute observation – nuclei in syncytia were undergoing division at different times, known as asynchronous division. She hypothesized that condensates are critical for creating different local environments around each nucleus so their division could be regulated independently. Dr. Gladfelter describes that, in this way, condensates could create different “neighborhoods” within syncytia that allow nuclei to regulate their cell cycles asynchronously. 

Dr. Gladfelter supports her hypothesis with her lab’s observations of Whi3, a fungal protein that binds an mRNA encoding the cell cycle-regulating protein cyclin. Dr. Gladfelter and her group have observed that fungal cells without Whi3 don’t form condensates, and these cells exhibit synchronous nuclear division. Based on this evidence, it appears that forming condensates with Whi3 protein and cyclin mRNA is essential for fungal syncytia to control how different nuclei are dividing at different times. 

Because these condensates are critical for regulation in fungal cells, Dr. Gladfelter wanted to understand factors that influence how Whi3 and cyclin mRNA form condensates and how they affect nuclear division. Her group collected fungal samples from regions across the U.S. with different climates. Comparing fungal isolates from Florida to isolates from Wisconsin, they observed asynchronous nuclear division when these cells were at temperatures similar to their natural environment. But when taken to temperature extremes compared to their origin (i.e., cold temperatures for Florida isolates and warm temperatures for Wisconsin isolates), the nuclei in these cells started dividing synchronously. 

But if fungi from Florida and Wisconsin are both regulating nuclear division using condensates formed by Whi3 protein and cyclin mRNA, why are these processes dependent on the temperature of their isolate origin? Dr. Gladfelter and her group have analyzed structures of Whi3 proteins and cyclin mRNAs for clues. Specifically, they wondered how the degree of order, or how organized and structured certain molecules are, impacts the interactions between molecules in condensates. Dr. Gladfelter and her lab hypothesized that because the number and degree of structured regions in a protein or RNA molecule can impact their ability to interact with other molecules, their structures might impact their ability to form condensates. 

Dr. Gladfelter and her lab first examined how order impacts protein constituents of condensates. They compared natural variation in Whi3 protein sequences in their collection of fungal isolates from different climates. Interestingly, they found seemingly small changes to these protein sequences depending on the isolate’s original climate, but these small changes resulted in big implications for condensate function. They found that Whi3 proteins had more disordered (or “looser knit”) structures in fungal isolates from cold climates compared to those from warm environments. This change has consequences for these cold-dwelling fungi, as having more disordered regions in Whi3 promotes phase separation to form condensates. Since condensate formation is critical for asynchronous nuclear division, this degree of disorder in Whi3 from cold isolates is likely a beneficial adaptation. 

To understand how structural changes in cyclin RNA impact condensates, Dr. Gladfelter and her group used computer modeling. In contrast to their approach looking for natural variation in Whi3 protein structures from their fungal isolates, her group used in silico evolution to test predicted structures of hypothetical, computer-generated cyclin RNAs. They made small changes to the nucleotide sequences that make up the cyclin RNA, without changing their overall composition or the protein sequence they encode. They then modeled the relative stability of these RNAs and observed how it influenced characteristics of the condensates. Through this analysis, they found that RNA with less structure formed condensates that are more elastic, age faster, and are more often associated with fungi from hot environments. This finding suggests that altering RNA structure is a way that fungi can optimize the properties of their condensates and adapt to the temperature of their environment. 

The degree of order in both protein and RNA structures that make up condensates have consequences for how they form and function because they influence their ability to interact with other molecules. The differences in condensate properties depending on the native climate of different fungi show an important role for condensates in helping fungi to adapt to their environment. Dr. Gladfelter concludes her talk stressing the timely implications of these temperature adaptations through condensates as we experience climate change. She also emphasized the need for future research to not be limited to the nucleotide sequence of RNA or the amino acid sequence of proteins as critical drivers for functions of these molecules. Rather, researchers must also continue to examine the impact of structure and order as additional facets of complexity that define interactions between molecules, because their effects are impactful to the cell’s biology. 

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About the speaker

Dr. Amy S. Gladfelter is a Professor of Biology at the University of North Carolina at Chapel Hill. She earned her B.A. in molecular biology from Princeton University and her Ph.D. in genetics at Duke University. After earning her Ph.D., Dr. Gladfelter was a postdoctoral fellow at the University of Basel Biozentrum. She joined the faculty in Biological Sciences at Dartmouth College before moving to the University of North Carolina at Chapel Hill in 2016. In 2021, Dr. Gladfelter was elected as an AAAS fellow. Her research interests center on cellular organization, including: phase transitions, cell shape and spatial sensing, and using marine fungi as a model system for cells surviving extreme environments. More information about Dr. Gladfelter and her work can be found on her website.