ORCID
https://orcid.org/0000-0002-7877-6285
Date of Award
11-16-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Biomolecular condensates form via the phase transitions of proteins and nucleic acids. What is unclear is how unique condensates achieve compositional specificity. Of particular interest is the ability of distinct condensates to have shared and distinct components. This is readily apparent in the multinucleate filamentous fungi Ashbya gossypii, where condensates share a common cytoplasm. In Ashbya, the RNA-binding protein Whi3 forms condensates with CLN3, BNI1, and SPA2. These RNAs are responsible for initiating the cell cycle, forming actin cables, and directing polarized cell growth respectively. However in vivo observations have shown that certain Whi3-RNA condensates are capable of mixing while others remain distinct, despite Whi3 being a common component of each condensate; Whi3-BNI1 condensates colocalize with Whi3- SPA2 condensates, whereas Whi3-CLN3 condensates do not colocalize with Whi3-BNI1 or Whi3- SPA2 condensates. Here I present findings that demonstrate how dynamical control establishes demixed and mixed condensates in vitro as well as in live Ashbya cells. We demonstrate in vitro how the order of operations with respect to RNA addition can engender mixed or demixed condensates, regardless of RNA identity. Analyses of Whi3-RNA phase boundaries reveal Whi3-RNA heterotypic interactions driving condensate formation, with temporal control of RNAs and RNA- RNA homotypic interactions contributing to the formation of demixed condensates in ternary mixtures. In a facsimile of our in vitro temporal control schema, we also demonstrate in vivo how perturbing wildtype expression patterns of RNAs that normally do not colocalize is sufficient for generating mixed condensates. We also observe measurable defects in fungal morphology, suggesting that dynamical control is necessary for proper cell growth. Over the course of this dissertation, I have developed a robust pipeline for the routine expression and fluorescent tagging of CLN3, BNI1, and SPA2 which cover a wide range of lengths and sequence complexity. The methods we have developed are applicable to other biologically- relevant RNAs and will be a useful addition to any RNA biochemistry toolbox. Finally, we demonstrate how the composition and patterning of the Whi3 glutamine-rich region is capable of modulating homotypic and heterotypic phase separation with BNI1 RNA. Collectively, our findings demonstrate how dynamical control is a likely mechanism for the demixing of condensates in vitro and in vivo, and how the composition and patterning of polar residues can affect homotypic and heterotypic phase behavior.
Language
English (en)
Chair and Committee
Rohit Pappu
Recommended Citation
Lin, Andrew Zijia, "Uncovering the Determinants of Compositionally Distinct, Coexisting Biomolecular Condensates" (2023). Arts & Sciences Electronic Theses and Dissertations. 3189.
https://openscholarship.wustl.edu/art_sci_etds/3189