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. Author manuscript; available in PMC: 2017 Jul 14.
Published in final edited form as: Cell. 2016 Jul 14;166(2):408–423. doi: 10.1016/j.cell.2016.06.037

Figure 4. Endosome-ER Tethering via an SNX2-VAPB Interaction.

Figure 4

(A) Confocal images of COS-7 cells expressing mCh-VAPB alone, or co-expressing YFP-SNX2 and mCh-VAPB, showing the enrichment of mCh-VAP at YFPSNX2-positive endosomes. Scale bar, 10 μm.

(B) Confocal images of a COS-7 cell co-expressing YFP-SNX2 and mCh-VAPB upon treatment with the VPS34 inhibitor VPS34-IN1 (1 μM). The dissociation of YFP-SNX2 from endosomes correlates with the dispersal of mCh-VAPB throughout the ER. Quantification is shown at right. Scale bar, 5 μm. See also Movie S2.

(C and D) Confocal images and line scan analysis of COS-7 cells overexpressing YFP-SNX1 and mCh-VAPB (C), or YFP-SNX2 and mCh-VAPB (D), showing that mCh-VAPB coclusters selectively with YFP-SNX2 but not with YFP-SNX1. Scale bar, 5 μm.

(E) Extracts of HeLa cells transfected with the constructs indicated were subjected to anti-GFP immunoprecipitation (IP) and then processed for SDS-PAGE and immunoblotting (IB) with anti-Myc or anti-HA antibodies.

(F and G) Confocal images and line scan analysis of COS-7 cells co-expressing YFP-SNX2 and either the FFAT motif-binding-deficient mutant mCh-VAPBKMDD (double mutant K87D M89D) (F), or the ALS8 mutant mCh-VAPBP56S (G), showing that mCherry fluorescence remains diffuse throughout the ER tubular network and does not cocluster with YFP-SNX2. Scale bar, 5 μm.

(H) Top: SNX2 domain structure. Note the presence of sequences containing phenylalanine residues and acidic amino acids. Bottom: extract of HeLa cells transfected with myc-VAPBWT and WT or mutant YFP-SNX2 were subjected to anti-GFP immunoprecipitation (IP) and then processed for SDS-PAGE and immunoblotting (IB) with anti-GFP and anti-Myc antibodies.

(I and J) Confocal images and line scan analysis of COS-7 cells co-expressing mCh-VAPB and either YFP-tagged N-terminal fragment of SNX2 (YFP-SNX21–139) (I), or YFP-SNX2F28A (J). YFP-SNX21–139, which lacks the endosome binding sites but contains the FFAT-like motif, colocalizes with VAPB throughout the ER. YFP-SNX2F28A localizes to endosomes but fails to cocluster with VAPB. Scale bar, 5 μm.

(K) Confocal images of COS-7 cells showing that OSBP-EGFP has a predominant diffuse localization when overexpressed alone, is recruited to the ER membrane when co-overexpressed with mCh-VAPB, and is also co-enriched with mCh-VAPB at iRFP-SNX2-positive hotspots when coexpressed with both these proteins. Scale bar, 5 μm.

See also Figure S4.