Dissertations, Theses, and Capstone Projects

Date of Degree

9-2023

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Shana Elbaum-Garfinkle

Committee Members

Rein Ulijn

Raymond Tu

Allie Obermeyer

Nicolas Biais

Subject Categories

Biochemistry | Biophysics | Structural Biology

Keywords

Neurodevelopment, phase separation, condensates, elastin, material properties

Abstract

The discovery of membraneless organelles has revolutionized the field of cell biology and our understanding of cell compartmentalization. These organelles lack a distinct phospholipid bilayer and interact with the cytoplasm mediated by a liquid-liquid interface. The emergent properties and characterization of these organelles are essential for decoding the mechanisms underlying their function and dysfunction. In this thesis, we focus on two reconstituted in-vitro model systems of protein condensates and advance current methodology in order to address these fundamental questions. The first model system examined is the neuronal granule protein Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein, that aids in the translocation and processing of mRNA in the neuron. The silencing of the gene encoding FMRP, results in the development of Fragile X Syndrome, one of the most common genetic causes of autism and intellectual disabilities. FMRP mediates translational repression and localization of mRNA. However, the binding specificity between the protein and its RNA targets is poorly understood. Previous studies suggest that the low complexity region (LCR) of FMRP has a preferential binding affinity towards G-quadruplex - containing mRNA. We find that these RNAs can modulate the material properties of FMRP-LCR liquid droplets as a function of secondary structure and binding affinity. Our findings lend insight into the mechanisms by which targeted RNA molecules modulate the material properties of neuronal granules. In order to gain more insight into material transitions, we next examined a model system based on tropoelastin, an extracellular protein relevant in connective tissue assemblies like lungs, arteries, and cartilage. The coacervation and cross-linking process of tropoelastin culminates in the formation of elastin fibers, a resilient biomaterial capable of withstanding numerous cycles of stress and strain. Tropoelastin can undergo liquid-liquid phase separation in vitro. This event aids in the self-assembly and subsequent maturation of elastin fibers. Although the mechanical properties of mature elastin fibers are well characterized, elastin phase separation and its transition to solid-like aggregates remain poorly understood. We use a model mini-elastin polypeptide to mimic the domain architecture of tropoelastin to measure this transition. We find that elastin droplets behave as viscous fluids at early incubation times, followed by a rapid liquid-to-solid transition as a function of time without an enzyme cross-linker. We further resolve the changes in dynamics, diffusion, and material properties of elastin condensates throughout this transition. This work, which reveals the material transition from within elastin condensates, lends new insight into the early steps of the self-assembly process of elastin while also contributing to the expanding repertoire of condensate maturation models in biological systems. Lastly, to expand on the suite of techniques available to study condensates, we develop a fluorescence microscopy assay to measure the density of condensates. Additionally, atomic force microscopy is used to measure their surface tension. This work provides insight into elements that determine the material properties of biomolecular condensates.

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