Shelby Stakenas
Pronouns: (She/They)
Research Mentor(s): Nils Walter
Research Mentor School/College/Department: Chemistry / LSA
Program: UROPF
Session: Session 5 (2:40pm – 3:30pm)
Authors: Shelby Stakenas, Emily Sumrall, Nils Walter
Abstract
Ribonucleoprotein (RNP) granules are membraneless organelles–termed condensates– formed through the process of liquid-liquid phase separation. RNP granules have many important functions within the cell including RNA metabolism (Tauber, D., et al, 2020). Fused in Sarcoma (FUS) is an intrinsically disordered, RNA-binding protein that regulates transcription. When found within the nucleus of a cell, FUS condensates readily bind to mRNA strands. The prion-like protein FUS causes a progression of neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) when mutated (Patel, A., et al, 2015). We have developed a method of reconstituting FUS condensates and tracking RNA diffusion that is sensitive enough to detect different diffusion states of mRNA as a function of physiochemical conditions. Using in-vitro transcription and click chemistry to label Firefly Luciferase (FL) mRNA, the mRNA can be used as a probe to track the diffusion of RNA within FUS condensates, visualized by single-molecule microscopy (Kolb, H. et al, 2001). Our microscope slides are treated with biotin-PEG which is used as a tether allowing streptavidin to link to biotinylated FUS protein to the surface. This provides a handy way to tether our condensates to the surface which is necessary for single-particle tracking. With an ONI Nanoimager, the HILO (Highly Inclined and Laminated Optical sheet) angle is used to penetrate the condensates to produce a 2-dimensional plane for visualizing the diffusion of the labeled mRNA within the FUS condensates. From these images, the mRNA trajectories can be quantified. Important information can be derived from tracking the labeled mRNA about the diffusion behavior of the RNA. The trajectories can be used to generate a MSD-tau plot, diffusion coefficients, and apparent D distribution. These experiments demonstrate how the use of a crowding agent, dextran, impacts the diffusion of FL mRNA. Our method also reveals how total RNA influences the diffusion characteristics of FL mRNA. When spinal cord total RNA is introduced into the experiment, the mRNA trajectory count decreases. The same trend is observed when dextran is present in addition to a visual decrease in FUS condensates. This is likely due to the constrained movement of the mRNA which excludes their trajectories from a normal diffusion model.
