Supplementary MaterialsSupplementary information joces-131-214692-s1. 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 liquidCliquid phase separation and their deregulation in human diseases. while they are potentially critical to trigger phase separation (Aguzzi and Altmeyer, 2016; Bounedjah et al., 2012). Therefore, there is a need to develop methods to probe phase separation in a cellular context (Banani et al., 2016; Patel et al., 2015; Shin et al., 2017). Here, we present a method to probe phase separation by confining selected RBPs on microtubules in fixed or living mammalian cells (Fig.?1A,B). There are three major advantages in using this method. First, the geometry of microtubules (micrometer-long cylinders with nanometer-size diameter) enables RBPs to be confined in order to detect and quantify their spatial segregation along microtubules. Other fluorescence methods, such as fluorescence resonance energy transfer (FRET) and complementation assays (Xing et al., 2016) detect interactions between two proteins, which is inappropriate to investigate phase separation as, for example, proteins that share the same compartment may not cause a FRET signal (no direct interaction). Second, the spatial separation of virtually any protein couple can be analyzed provided that they can be brought onto microtubules and irrespective of their solubility, which is a major concern for investigations. Third, the compartmentalization of truncated or mutated proteins confined on microtubules can be visualized and measured, whereas protein truncation or mutation will often change the location of proteins from their original compartments, which would hinder studies LP-533401 cost on the structural basis of sub-compartmentalization in cells. Open in a separate window Fig. 1. RBPs are confined LP-533401 cost into compartments along the microtubule network after their fusion to tau. (A) Schematic of the method used to bring RBPs on microtubules through their fusion to tau, a microtubule-associated protein. (B) All tauCRFPCRBPs tested (FUS, G3BP1, HuR and TDP-43) were brought onto microtubules in HeLa cells. tauCRFPCRBP is shown in red and anti–tubulin staining in green. Scale bar: 10?m. (C) Images of Hela cells co-expressing tauCRFP and either tauCGFP and tauCGFPCTDP-43. Note the spatial segregation on microtubules induced by the fusion of TDP-43 to tauCRFP. Scale bar: 10?m. (D) Time-lapse images (time in minutes) of tauCGFPCTDP-43. Note the fusion of two tauCGFPCTDP-43 compartments moving along a microtubule (see arrows). Scale bar: 1?m. In this article, we analyzed whether four mRNA-binding proteins, TDP-43, FUS, HuR (also known as ELAVL1) Rabbit Polyclonal to RPAB1 and G3BP1 could form liquid phases when confined on microtubules. TDP-43 and FUS are known to form liquid droplets due to their self-attracting low complexity domains (LCDs) (Gopal et al., 2017; Murakami et al., 2015; Murray et al., 2017; Patel et al., 2015; Uversky, 2017). HuR (Aulas et al., 2015; Fialcowitz-White et al., 2007; Kedersha et al., 2016) and G3BP1 (Abrakhi et al., 2017) do not display established self-attracting LCDs. We found that confining any of these RBPs, and thus mRNAs, on microtubules leads to the formation of mRNA-rich liquid-like compartments on microtubules, irrespective of the LCD presence. To demonstrate the usefulness of our approach, the miscibility between different mRNA-rich compartments formed by bringing two different RBPs on microtubules was analyzed. We then focused our analysis on the roles of the RNA-binding domain (RBD) and LP-533401 cost the LCD in the mixing and demixing between coexisting compartments. RESULTS Confining RBPs on microtubules does not prevent their binding to mRNA and leads to the formation of RBP compartments To confine RBPs on microtubules, they were fused to tau (also known as MAPT) (Boca et al., 2015) (Fig.?1A,B), a microtubule-associated protein, labeled with either RFP or GFP. Tau has a higher affinity.