Oral Presentation The 45th Lorne Conference on Protein Structure and Function 2020

ALS-associated Mutations in the TDP-43 Low-Complexity Domain have Variable Effects on its Liquid-Liquid Phase Separation Properties (#47)

Luke McAlary 1 2 , Tiam Heydari 1 , Johnathan Hoggarth 1 , Xubiao Peng 1 , Mine Sher 1 2 , Zoe Gidden 1 2 , Neil R Cashman 2 , Steven S Plotkin 1
  1. Department of Physics and Astronomy, University of British Columbia, Vancouver, British Columbia, Canada
  2. Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada

The C-terminal low-complexity domain (LCD) of TDP-43 has been shown to mediate TDP-43's ability to undergo liquid-liquid phase separation (LLPS) and form biomolecular condensates[1]. ALS/FTD mutations are localized to this region, suggesting that mutations may alter the LLPS characteristics of the protein. Multiple studies have highlighted the impact of de novo mutations on TDP-43's LLPS characteristics, yet few studies have investigated the role of ALS-associated mutations. Therefore, we have examined the effects of multiple ALS mutations on the LLPS characteristics of TDP-43.

TDP-43 LCD (267-414) mutants were examined bioinformatically using several sequence-based tools that focused on biophysical attributes. Multiple-criteria decision analysis (MCDA) was employed to predict the LLPS propensity of mutants.

14 ALS mutant TDP-43 LCD variants were chosen from MCDA and purified from E coli. LLPS characteristics were examined via microscopy, turbidimetry, and centrifugation. Amyloid characteristics were examined using Thioflavin-T, semi-denaturing PAGE, and TEM.

In-cell experiments were performed using an optogenetic system[2]. U2OS cells were transiently transfected with optoTDP-LCD variants and LLPS characteristics were quantified by examining the kinetics of optodroplet formation.

MCDA predicted both LLPS promoting mutants and LLPS deficient mutants on the basis of weighting for or against the physical properties associated with LLPS. We chose variants from each category to examine further, finding that the predictions generally matched LLPS characteristics in vitro. 

We next examined the tendency of the LCD variants to form amyloid-like structures. Unfavourable LLPS conditions (low salt) were found to promote amyloid formation whereas LLPS promoting conditions (high salt) were found to impede the formation of amyloid-like assemblies.

Cellular examination of variants using an optogenetic system revealed that the in vitro-determined LLPS properties did not always correlate with the cellular measurements. Determination of the saturation concentration of mutants showed that several LLPS deficient mutants had similar in-cell LLPS characteristics to WT.

Since most ALS-associated mutations in TDP-43 occur within its LCD, it is reasonable to hypothesize that they would systematically alter LLPS properties. The results presented here suggest however that these mutations can either increase or decrease LLPS propensity. This result is important for understanding the relationship between aberrant biological LLPS and ALS.

  1. [1] Conicella AE, Zerze GH, Mittal J et al Structure 2016; 24:1537-1549
  2. [2] Shin Y, Berry J, Pannucci N, et al Cell 2017; 168: 159-171