Kai Littlejohn, B.S.
BME PhD Proposal Presentation
Date: 2024-11-18
Time: 1:00-3:00pm
Location / Meeting Link: HSRB I-E160
Committee Members:
Felipe Quiroz, PhD (Advisor) Levi Wood, PhD David Lynn, PhD Karmella Haynes, PhD Anant Paravastu, PhD
Title: Biomolecular tools for live-cell imaging of disordered proteins and their neuropathological assemblies
Abstract:
Intrinsically disordered proteins (IDPs) have emerged as key drivers of cell physiology and neuropathology. IDPs and IDP domains, across eukaryotic proteomes, exploit their heterogeneous conformational dynamics to fuel the self-assembly of intracellular and extracellular structures. Select IDPs undergo intracellular liquid-liquid phase separation (LLPS), reversibly de-mixing into two liquid phases: an IDP-rich biomolecular condensate (BMC) and a surrounding dilute aqueous phase. Condensate-forming IDPs function as scaffolds that recruit additional biomolecules to orchestrate cellular processes. The hallmark of many neurodegenerative diseases, including Alzheimer’s Disease (AD) and Amyotrophic lateral sclerosis (ALS), is the accumulation of intracellular IDP aggregates. Recent in vitro studies have suggested that neuropathological IDP aggregates begin as liquid-like BMCs that mature into solid-like IDP-assemblies. Unfortunately, the live-cell probing of IDPs and their intraneuronal self-assembly dynamics remains challenging, particularly in neurodegeneration-relevant contexts. To this end, our group recently introduced genetically-encoded LLPS-sensors for innocuous quantitative live-cell probing of epidermal BMCs that are key to skin barrier formation. Translating this concept and progress to the live-cell examination of IDP-linked neuropathology, this proposal will engineer and test LLPS-sensors directed to TDP43-assemblies, which feature prominently in AD and ALS brains. Because extant live-cell technologies rely on molecular-level tagging with fluorescent proteins, Aim 1 will quantitatively characterize the impact of fluorescent protein tags on the LLPS behavior of diverse IDPs, including TDP43, informing choice of future IDP tags and exposing the need for alternative live-cell biomolecular tools. For LLPS-sensor evolution, Aim 2 will demonstrate and exploit a novel intracellular bacterial platform to readily access TDP43-assemblies that are otherwise unique to aging and stressed neurons. Using protein engineering and quantitative live-cell microscopy, this work will generate candidate LLPS-sensors for sensitive probing of TDP43-assemblies. Finally, Aim 3 will test high-performing LLPS-sensors from Aim 2 in a disease-relevant neuronal model of ALS. The evolution and application of tunable LLPS-sensitive tools to intraneuronal TDP43-sasemblies will provide a strong foundation for engineering an array of intracellular LLPS-sensors directed to neuropathological IDP-assemblies. More broadly, the results of this work will stimulate the engineering of biomolecular tools for innocuous live-cell probing of IDPs and their physiological and pathological BMCs.
