Open AccessMicrotubules as platforms for probing liquid-liquid phase separation in cells: application to RNA-binding proteins
Author(s) -
Alexandre Maucuer,
Bénédicte Desforges,
Vandana Joshi,
Mirela Boca,
Dmitry A. Kretov,
Loïc Hamon,
Ahmed Bouhss,
Patrick A. Curmi,
David Pastré
Publication year - 2018
Publication title -
journal of cell science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.384
H-Index - 278
eISSN - 1477-9137
pISSN - 0021-9533
DOI - 10.1242/jcs.214692
Subject(s) - compartmentalization (fire protection) , microtubule , biology , rna , messenger rna , stress granule , context (archaeology) , rna binding protein , microbiology and biotechnology , p bodies , biophysics , biochemistry , translation (biology) , gene , enzyme , paleontology
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.