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

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.

Role of tau in the spatial organization of axonal microtubules: keeping parallel microtubules evenly distributed despite macromolecular crowding.

Opposing views have been proposed regarding the role of tau, the principal microtubule-associated protein in axons. On the one hand, tau forms cross-bridges at the interface between microtubules and induces microtubule bundling in neurons. On the other hand, tau is also considered a polymer brush which efficiently separates microtubules. In mature axons, microtubules are indeed arranged in parallel arrays and are well separated from each other. To reconcile these views, we developed a mechanistic model based on in vitro and cellular approaches combined to analytical and numerical analyses. The results indicate that tau forms long-range cross-bridges between microtubules under macromolecular crowding conditions. Tau cross-bridges prevent the redistribution of tau away from the interface between microtubules, which would have occurred in the polymer brush model. Consequently, the short-range attractive force between microtubules induced by macromolecular crowding is avoided and thus microtubules remain well separated from each other. Interestingly, in this unified model, tau diffusion on microtubules enables to keep microtubules evenly distributed in axonal sections at low tau levels.

Probing protein interactions in living mammalian cells on a microtubule bench.

Microtubules are µm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases.

Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants.

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.

SOD1 and amyotrophic lateral sclerosis: mutations and oligomerization.

There are about 100 single point mutations of copper, zinc superoxide dismutase 1 (SOD1) which are reported (http://alsod.iop.kcl.ac.uk/Als/index.aspx) to be related to the familial form (fALS) of amyotrophic lateral sclerosis (ALS). These mutations are spread all over the protein. It is well documented that fALS produces protein aggregates in the motor neurons of fALS patients, which have been found to be associated to mitochondria. We selected eleven SOD1 mutants, most of them reported as pathological, and characterized them investigating their propensity to aggregation using different techniques, from circular dichroism spectra to ThT-binding fluorescence, size-exclusion chromatography and light scattering spectroscopy. We show here that these eleven SOD1 mutants, only when they are in the metal-free form, undergo the same general mechanism of oligomerization as found for the WT metal-free protein. The rates of oligomerization are different but eventually they give rise to the same type of soluble oligomeric species. These oligomers are formed through oxidation of the two free cysteines of SOD1 (6 and 111) and stabilized by hydrogen bonds, between beta strands, thus forming amyloid-like structures. SOD1 enters the mitochondria as demetallated and mitochondria are loci where oxidative stress may easily occur. The soluble oligomeric species, formed by the apo form of both WT SOD1 and its mutants through an oxidative process, might represent the precursor toxic species, whose existence would also suggest a common mechanism for ALS and fALS. The mechanism here proposed for SOD1 mutant oligomerization is absolutely general and it provides a common unique picture for the behaviors of the many SOD1 mutants, of different nature and distributed all over the protein.