Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies

Abstract

Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport. Under conditions where FUS phase separates into liquid droplets in vitro, the addition of Nup62 caused the synergistic formation of amorphous assemblies containing both FUS and Nup62. Here, we examined the molecular determinants of this process using recombinant FUS and Nup62 proteins and biochemical approaches. We demonstrate that the structured C-terminal domain of Nup62 containing an alpha-helical coiled-coil region plays a dominant role in binding FUS and is sufficient for inducing the formation of FUS/Nup62 amorphous assemblies. In contrast, the natively unstructured, F/G repeat-rich N-terminal domain of Nup62 modestly contributed to FUS/Nup62 phase separation behavior. Expression of individual Nup62 domain constructs in human cells confirmed that the Nup62 C-terminal domain is essential for localization of the protein to the nuclear envelope. Our results raise the possibility that interactions between FUS and the C-terminal domain of Nup62 can influence the function of Nup62 under physiological and/or pathological conditions.

Keywords: amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); fused in sarcoma (FUS); nucleocytoplasmic transport; nucleoporin 62 (Nup62); phase separation.

Figure 1.. Robust binding occurs between the C-terminal domain of Nup62 and FUS.

(A) Schematic representation of the Nup62 domain architecture (top) and Nup62 disorder propensity as predicted by DISOPRED3 (59,60) (bottom). Nup62 has a disordered N-terminal domain (NTD, purple) containing 23 phenylalanine residues (red) and a C-terminal domain (CTD, orange) with an alpha-helical coiled coil region (yellow; domain boundary determined using Pfam (61)). (B) Western blot for the GST-pull down assay probed with anti-MBP for detection of Nup62 and anti-FUS for detection of FUS. GST-FUS was incubated with the indicated MBP-Nup62 construct (Input, left). Complexes were pulled-down with glutathione beads (right). (C) As in (B), except GST (left blot) and MBP (right blot) are used as negative controls. Results are representative of n=3 independent experiments.

Figure 2.. The C-terminal domain of Nup62 is sufficient for the formation of amorphous assemblies with FUS.

(A) Fluorescence images showing phase separation of MBP-FUS (red) with MBP-Nup62 domain constructs (green). MBP-FUS was doped with <5% of Alexa-555 labelled MBP-FUS and MBP-Nup62 domains were doped with <5% of the corresponding Alexa-488 labelled MBP-Nup62 construct. MBP-FUS assembles into spherical droplets but forms amorphous assemblies in the presence of MBP-Nup62 full length (FL) and MBP-Nup62 CTD. Representative images from at least n=3 independent experiments are shown. Scale bars 50 μm or 5 μm (inset). (B) Quantification of reactions with amorphous assemblies shown in (A). The image area occupied by amorphous assemblies were normalized to reactions containing MBP-FUS FL + MBP-Nup62 FL for each experiment. Each point represents the average of at least 3 fields of view (FOV) in an experiment. Bars depict mean ± SD. Paired t-test with comparison to MBP-FUS FL + MBP-Nup62 FL; *p<0.05. (C) Quantification of droplet size and abundance (D) for the indicated reactions from (A) and (E). Each point represents the average of at least 3 fields of view (FOV) in an experiment. Bars depict mean ± SD. Paired t-test with comparison to MBP-FUS droplets; **p<0.01, ****p<0.0001. Only statistically significant comparisons are shown. (E) Fluorescence images showing phase separation of MBP-FUS FL (red) with MBP-Nup62 NTD F->A (green). Representative images from at least n=3 experiments are shown. Scale bars 50 μm or 5 μm (inset). See panels C and D for quantification.

Figure 3.. Nup62 CTD localizes to the nuclear envelope.

(A) The C-terminal domain of Nup62 (magenta) is shown embedded within the structure of the inner ring of the nuclear pore complex (PDB ID: 5IJN) (43,62). Mol* was used to create (A) (63). (B) Immunofluorescence imaging of semi-permeabilized ReNcell VM cells expressing mApple or mApple-tagged Nup62 full length (FL), NTD and CTD probed with anti-mApple (red) to amplify the mApple fluorescence signal and anti-POM121 (yellow) to mark the nuclear envelope. (C) As in (B) except images are acquired with longer exposure time (denoted by “HI” in the micrograph) for detection of anti-mApple signal. Images are representative of n=3 biological experiments. Scale bars 22.5 μm or 7.5 μm (inset)

Figure 4.. The FUS RGG3 domain is sufficient whereas as the QGSY domain is dispensable for FUS binding to Nup62.

(A) Schematic representation of the domain architecture of FUS (top) and the disorder propensity predicted by DISOPRED3 (59,60) (bottom). FUS contains an N-terminal QGSY-rich domain, three RGG domains, a structured RNA recognition motif (RRM) and zinc finger (ZnF) motif. In addition, FUS encodes a nuclear localization signal (NLS) and a predicted nuclear export signal (NES). (B) FUS constructs used in this study. (C) Western blot of co-immunoprecipitation (IP) reactions wherein MBP-Nup62 FL was pulled down from reactions containing the indicated MBP-FUS domain constructs (Left). Western blot of co-IP reactions lacking MBP-Nup62 FL (Right). Results are representative of n=2 experiments.

Figure 5.. Analysis of co-phase separation behavior for FUS domain constructs in the presence of Nup62.

(A) Fluorescence images showing phase separation of MBP-FUS domain constructs doped with <2% of the corresponding Alexa-555 labelled proteins. Under the same conditions as MBP-FUS full length (FL), only MBP-FUS ΔQGSY formed droplets. The exposure and brightness settings of the images denoted by “HI” were increased to better visualize the presence (or absence) of assemblies. Representative images from n=3 experiments are shown. Scale bars 50 μm or 5 μm (inset). (B) MBP-FUS ΔQGSY droplets are smaller in size and (C) fewer in number as compared to MBP-FUS FL droplets. Each point represents the average of at least three fields of view (FOV) in an experiment. Bars depict mean ± SD. Paired t-test. ***p<0.001, ****p<0.0001. (D) As in (A) but mixed with Nup62. Representative images from n=3 experiments are shown. Scale bars 50 μm or 5 μm (inset). (E) Formation of amorphous assemblies is reduced in reactions of MBP-FUS ΔQGSY + MBP-Nup62. Each point represents the average among three fields of view (FOV) in an experiment. Bars depict mean ± SD. Paired t test. ****p<0.0001.

Figure 6.. Charge plot of FUS and Nup62.

(A) The C-terminal regions of FUS including RGG domains and (B) the C-terminal domain of Nup62 are enriched in positively and negatively charged residues, respectively. Plot generated using EMBOSS (64).

Figure 7.. Salt concentration modulates the formation of FUS/Nup62 amorphous assemblies.

Fluorescence images showing phase separation of MBP-FUS (red) doped with <5% of Alexa-555 labelled MBP-FUS and MBP-Nup62 (green) doped with <5% of Alexa-488 labelled MBP-Nup62 in the presence of varying concentrations of sodium chloride. The formation of MBP-FUS droplets and MBP-FUS/MBP-Nup62 amorphous assemblies is reduced at high salt concentration. Representative images from n=2 experiments are shown. Scale bars 50 μm or 5 μm (inset).