b Immunostaining of 4?month old spleen sections (red pulp region) of WT and C9orf72-/- mice with anti-mouse CD68, prosaposin (PSAP), and progranulin (PGRN) antibodies. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0324-5) contains supplementary material, which is available to authorized users. test. em P /em -values 0.05 were considered statistically significant. Results C9orf72 forms a complex with SMCR8 and WDR41 In humans, two C9orf72 protein isoforms are generated from three alternatively spliced transcripts, a long form (C9-L) and a short form (C9-S), with multiple studies showing that the protein and mRNA level of the C9-L form are decreased in C9/ALS patients [5, 50, 52]. To decipher the protein interaction network of C9orf72, a SILAC Voriconazole (Vfend) (stable isotope labeling of amino acids in cell Voriconazole (Vfend) culture) based proteomic screen was performed in the neuroblastoma cells line neuro-2a (N2a) using GFP-C9orf72 (C9-L) as the bait and GFP as a control (Fig.?1a, Additional file 1: Figure S1). Several proteins were found to be enriched in the C9orf72 immunoprecipitations (IPs) (Fig.?1b, Additional file 2: Table S1). The top two hits from the screen were SMCR8 and WDR41, two proteins of unknown functions (Fig.?1b). Interestingly, like C9orf72, SMCR8 was also predicted to contain a DENN domain [54]. The interaction between C9orf72, SMCR8, and WDR41 was verified using co-IPs in transfected HEK293T cells (Fig.?2a-c). SMCR8 strongly interacts with GFP-C9orf72 but not GFP in the co-IP experiment (Fig.?2a). Moreover, co-expression of C9orf72 consistently increases the level of SMCR8, suggesting that C9orf72 might stabilize overexpressed SMCR8. However, the short isoform of human C9orf72 (C9-S) does not bind SMCR8 (Fig.?2a). Thus we focus on the C9-L form for the rest of the study (hereafter referred to as C9orf72). While we failed to detect any interaction between WDR41 and C9orf72 or SMCR8 when WDR41 is expressed with either C9orf72 or SMCR8 alone, WDR41 strongly co-immunoprecipitates with C9orf72 and SMCR8 when C9orf72 and SMCR8 are co-expressed, suggesting that WDR41 interacts only with the C9orf72/SMCR8 heterodimer Voriconazole (Vfend) (Fig.?2b, 2c). Open in a separate window Fig. 1 SILAC proteomic screen for C9orf72 binding partners. a Schematic workflow of SILAC proteomic screen ACAD9 used to identify C9orf72 protein interactions. b Volcano plot of SILAC hits. Hits with more than 10 peptides are plotted. Top hits identified in the heavy fraction are highlighted Open in a separate window Fig. 2 Co-immunoprecipitation between C9orf72, SMCR8 and WDR41. a GFP-tagged human C9orf72 isoform I (GFP-C9-L) or isoform II (GFP-C9-S) were overexpressed with SMCR8-myc in HEK293T cells and immunoprecipitated by anti-GFP beads. b SMCR8-GFP and WDR41-myc were coexpressed with or without FLAG-C9-L and the lysates were immunoprecipitated using anti-GFP antibodies. c GFP-C9-L and FLAG-WDR41 were co-expressed with or without SMCR8-myc as indicated and the lysates were immunoprecipitated using anti-GFP antibodies Cellular localization of C9orf72, SMCR8 and WDR41 To gain insight into the cellular function of the C9orf72/SMCR8/WDR41 complex, we expressed these proteins in HeLa cells and examined their distribution within the cell. Both C9orf72 and SMCR8 show diffuse cytoplasmic localization, when expressed alone or together (Fig.?3a and b). Nuclear localization was observed for C9orf72, especially the GFP-tagged C9orf72 but not SMCR8 (Fig.?3a and b). WDR41 also shows diffuse cytoplasmic distribution (Fig.?3a and ?andc).c). However, careful examination reveals enrichment of WDR41 at the cis-Golgi, which is confirmed when labelled by the cis-Golgi protein GPP130 (Fig.?3c). This is further supported by treatment of cells with BrefeldinA, which causes the Golgi to collapse. Even after such treatment, WDR41 remains colocalized with GPP130, indicating that WDR41 is tightly associated with the Golgi membrane. Similar results were obtained after treatment with nocodazole, a microtubule destabilizing drug that causes the Golgi to disperse (Fig.?3c). Open in a separate window Fig. 3 Cellular localization of C9orf72, SMCR8 and WDR41. a HeLa cells were transfected with FLAG-C9orf72 (C9-L), SMCR8-myc or WDR41-GFP. Cells were stained with anti-FLAG or anti-myc to visualize FLAG-C9orf72 or Voriconazole (Vfend) SMCR8-myc, respectively. Maximum projection images from confocal sections are shown. Scale bar?=?10?m. b HeLa cells were transfected with GFP-C9orf72?(C9-L) and SMCR8-myc. Cells were stained with anti-myc antibodies to visualize SMCR8-myc. c WDR41-GFP expressing HeLa cells were treated with DMSO control, 0.3?mM BrefeldinA (BFA), or 20?mM Nocodazole for 2?h. Cells were stained with anti-GPP130 antibodies to.