Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

Alexandre Maucuer (1) , Bénédicte Desforges (1) , Vandana Joshi (1) , Mirela Boca (1) , Dmitry Kretov (1, 2, 3) , Loic Hamon (1) , Ahmed Bouhss (1) , Patrick A. Curmi (1) , David Pastre (1)

1 SABNP - Structure et activité des biomolécules normales et pathologiques
2 Institute of Protein Research, Russian Academy of Sciences, Pushchino
3 BUSM - Boston University School of Medicine

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.

Keywords: Amyotrophic lateral sclerosis; Cellular compartment; Intrinsically disordered regions; Stress granules

Domains: Life Sciences [q-bio]

Alexandre Maucuer, Bénédicte Desforges, Vandana Joshi, Mirela Boca, Dmitry Kretov, et al.. Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins. Journal of Cell Science, 2018, 131 (11), pp.jcs214692. ⟨10.1242/jcs.214692⟩. ⟨hal-02166855⟩