Abstract
The study of RNA-binding proteins (RBP) offers insight into the mechanisms of pathologic protein aggregation in neurodegenerative diseases. We developed a protocol for purifying an RBP FUS and a nuclear import receptor (NIR) Kapβ2 and testing the ability of Kapβ2 to mitigate FUS aggregation and liquid-liquid phase separation.
Keywords: FUS, Liquid-liquid phase separation, Nuclear import receptor, Kapβ2, Protein aggregation
Background
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the mislocalization and aggregation of several RNA-binding proteins (RBP) (Mackenzie et al., 2010; Da Cruz and Cleveland, 2011; King et al., 2012). One of these proteins is FUS (Fused in Sarcoma), a nuclear protein mislocalizes in the cytoplasm of neurons in ALS/FTD patients, where it undergoes liquid-liquid phase transition followed by aberrant phase transition to form insoluble aggregates (Altmeyer et al., 2015; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015) (Figure 1). Karyopherin-β2 (Kapβ2), a nuclear import receptor (NIR), has been established as a chaperone and disaggregase of proteins with PY-nuclear localization sequences (NLS), such as FUS (Guo et al., 2018; Hofweber et al., 2018; Yoshizawa, et al., 2018). The following protocol was developed to test the ability of Kapβ2 to inhibit and reverse FUS aggregation and phase separation ex vivo (Guo et al., 2018).
Figure 1. Aberrant phase transition of FUS (10 μM) leads to the formation of fibrillar hydrogel. A. Domain architecture of FUS. B. DIC images show that FUS undergoes reversible liquid-liquid phase transition to form liquid droplets followed by aberrant phase transition to form hydrogels. Scale bar: 10 μm.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Analysis of FUS Aggregation
Recipes
Acknowledgments
This protocol was described briefly in Guo et al., 2018. This study was supported by grants from the NIH R21NS090205 (to J. Shorter), the G. Harold and Leila Y. Mathers Charitable Foundation (to J. Shorter), Target ALS (to J. Shorter), ALSA (to J. Shorter), and the Packard Center for ALS Research (to J. Shorter). We also acknowledge the Alzheimer's Association (AARF-16-441196) and Target ALS Springboard Fellowship to L.G.
Competing interests
The Authors declare that there is no conflict of interest.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.