Fig 2: GRAF2 and WDR44 are involved in the specific export of neosynthesized E-cadherin, MMP14, and CFTR ΔF508. (A) Confocal stacks of transfected HeLa cells stained with α–E-cadherin under nonpermeabilizing conditions or with α-MICAL1 under permeabilizing conditions. Upon cotransfection of all the dominant negative constructs, E-cadherin–GFP became trapped in intracellular compartments. E-cadherin–GFP colocalized with untagged MICAL1 (uMICAL1) G3W puncta and RFP–WDR44 ΔC patches. (B) Confocal stacks of transfected HeLa cells stained, in the case of uMICAL1 G3W, with α-MICAL1. Occludin-GFP was transported to the plasma membrane in all cases. Occludin-GFP was found in uMICAL1 G3W puncta, but not in WDR44 ΔC patches. (C) Biotinylation of surface proteins of shRNA-expressing HeLa cells transfected with E-cadherin–GFP and Occludin-GFP. The Transferrin receptor (TfnR) was used as loading control; it was also found as a dimer. The four parts of the blots are from the same membranes and correspond to identical exposure times. (D) Western blot analysis of an equal protein amount of shRNA-expressing HeLa cell lysates. The intensity of the Grp78 protein band was normalized by the Actin signal and expressed as a ratio to the value obtained for shControl-transfected cells. n = 2. Down-regulation of GRAF2 or WDR44 expression did not lead to an increase in Grp78 expression, a marker of ER stress (Lee, 2005). (E) Percentage of transfected HeLa cells, incubated or not with BFA (5 µg/ml, 15 min), with endogenous (endo.) WDR44 tubules. n = 24–31 for GFP-transfected cells and 4–6 for the others. Unlike under resting conditions (Fig. 4 D and Fig. 5 F), Rab10 T23N and MICAL1 G3W did not inhibit BFA-induced WDR44 tubules, suggesting that these may not be functional for protein export. (F) Confocal images of transfected HeLa cells, left untreated or incubated with biotin (40 µM, 1 h) and stained with α-Calnexin. In untreated cells, SBP-GFP–E-cadherin was in the ER and was released to the plasma membrane upon incubation with biotin. (D and E) Data are means ± SEM; **, P < 0.01; and ****, P < 0.0001. (A, B, and F) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

Fig 3: GRAF1b/2 colocalize with MICAL1 and WDR44. (A) Confocal stacks of transfected HeLa cells showing a similar distribution of N- and C-terminal–tagged MICAL1 and WDR44 (compare with Fig. 2 G). In some cells, WDR44 was found on irregular peripheral patches. (B) Confocal images of transfected HeLa cells showing GRAF1b-RFP on the same intracellular tubules as GFP-MICAL1. (C) Confocal images of transfected HeLa cells showing that although MICAL1 PPAPP did not efficiently colocalize with GRAF1b/2 (Fig. 2 I), it was sometimes found on GRAF-positive tubules (boxed areas). (D) Confocal images of transfected HeLa cells stained with α-MICAL1 and α-WDR44. Endogenous WDR44 and to a lesser extent endogenous MICAL1 colocalized with GRAF1b-GFP. (E) Confocal stacks of hTERT-RPE1, 293T, HeLa, U-87 MG, HT 1080, and COS-7 cells stained with α-WDR44. Endogenous WDR44 tubules of various lengths were found in all but hTERT-RPE1 cells. Tubules were more abundant and longer in HeLa, HT 1080, and U-87 MG cells. (F) Confocal images of transfected HeLa cells showing colocalization of GRAF1b/2 with WDR44 tubules but not with its peripheral patches. (G) Confocal images of transfected HeLa cells showing that WDR44 ΔPro was not recruited to GRAF1b/2 tubules. (H) Western blot analysis of shRNA-transfected HeLa cell lysates. Calnexin was used as loading control. Left and middle: Specific knockdown of WDR44 in cells expressing the shRNA-enconding plasmids shWDR44a and shWDR44b and of MICAL1 in cells expressing shMICAL1. The two parts of the left blots are from the same membrane and correspond to identical exposure times. Right: Immunoprecipitation (IP) of endogenous GRAF2 from equal amounts of cell lysates and Western blot analysis, showing specific knockdown of GRAF2 in shGRAF2a-transfected cells. The three parts of the blots are from the same membranes and correspond to identical exposure times. (I) Confocal images of shGRAF2a-expressing HeLa cells. The shGRAF2a-resistant protein GRAF2res-GFP and RFP-WDR44 colocalized on intracellular tubules; GRAF2res ΔBAR–GFP was diffuse and led to RFP-WDR44 being only found on puncta. (A–G and I) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

Fig 4: GRAF1b and GRAF2 bind to MICAL1 and WDR44. (A) Pull-down of HeLa cell proteins by GST, GST–GRAF1 SH3, or GST–GRAF2 SH3, analyzed on two replicate membranes. One was probed with α-WDR44, the other with α-MICAL1. They were then stained with Coomassie, revealing GST-tagged proteins. (B) Schematic representation of human GRAF2, MICAL1, and WDR44 and of mutants used. GRAF2 has two membrane-binding domains, BAR and PH, a GTPase Activating Protein (GAP) domain, and an SH3 domain. MICAL1 has an NADPH-binding MO domain, CH and LIM domains, a proline-rich region with a PPKPP motif, and a C-terminal coiled coil (CC). * indicates K832 mutated in MICAL1 PPAPP. WDR44 has an N-terminal FFAT motif, a proline-rich region, and seven WD repeats. (C and D) Immunoprecipitation (IP) of transfected 293T cells with α-GFP (C) or using α-myc–coated beads (D). The efficiency of GRAF2 binding (untagged GRAF2 [uGRAF2]; C) or GRAF2-GFP (D) was quantified, using coimmunoprecipitation with GFP-WDR44 (C) or myc-MICAL1 (D) as reference. n = 3 or 4. Binding was decreased by removal (C) or mutation (D) of proline-rich regions. (C) Note that GFP-WDR44 underwent proteolysis. (E) Endogenous WDR44 and GRAF2 were coimmunoprecipitated from a HeLa cell lysate using α-GRAF2 and α-WDR44, but not without antibody (−). The membrane was probed with α-GRAF2, followed by α-WDR44. The left and right parts of the membrane were treated identically. (F) Endogenous MICAL1 was coimmunoprecipitated from an SH-SY5Y cell lysate using α-GRAF2, but not without antibody (−). The membrane was probed with α-GRAF2, followed by α-MICAL1. (G) Confocal stacks of transfected HeLa cells. GFP-tagged GRAF1b, GRAF2, MICAL1, and WDR44 were cytosolic and associated with intracellular tubules. (H) Confocal images of transfected HeLa cells. GRAF2-RFP colocalized with GFP-tagged MICAL1 and WDR44. (I) Manders colocalization coefficients for GFP-tagged MICAL1 and WDR44 proteins on GRAF1b/2-RFP–associated structures. n = 15–30 cells. (J–L) Confocal images of HeLa cells stained with α-MICAL1 and α-WDR44 (J and K) or α-GRAF2 (L). (J) Untransfected cells had distinct WDR44 tubules but showed a diffuse distribution of MICAL1. (K) Endogenous WDR44 and to a lesser extent endogenous MICAL1 colocalized with GRAF2-GFP. (L) Endogenous GRAF2 was found on GFP-WDR44 tubules. (M and N) Percentage of transfected HeLa cells with endogenous (endo.) WDR44 tubules. (M) n = 4–8. (N) n = 3–8. (O) Confocal images of transfected HeLa cells stained with α-WDR44 and fluorescence intensity profiles along the tubule enlarged in the boxed area showing complementary distribution of untagged MICAL1 (uMICAL1, cyan line) and endogenous WDR44 (red line) on the same tubule. (C, D, I, M, and N) Data are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (G, H, J–L, and O) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

Fig 5: WDR44 tubules are in close contact with the ER via binding to VAPA/B. (A) Immunoprecipitation (IP) of endogenous proteins from 293T cells, untransfected or expressing myc-WDR44, with α-myc. Bound proteins were analyzed by electrophoresis and Coomassie staining. VAPA (Uniprot accession no. Q9P0L0) was identified by LC-MS/MS. (B) Confocal images of transfected HeLa cells showing GFP-VAPA or GFP-VAPB in reticular ER. (C) Immunoprecipitation (IP) of transfected 293T cells with α-GFP. myc-VAPB was coimmunoprecipitated by GFP-WDR44 but not by mutants lacking the first 14 aa of the protein. (D) Confocal images of transfected HeLa cells showing colocalization of WDR44 and VAPA/B on tubules and on peripheral patches. (E) Confocal images of transfected HeLa cells stained with α-VAPB showing colocalization of GFP-WDR44 patches and endogenous VAPB. (F) Confocal images of transfected HeLa cells stained with α-myc and α-Calnexin or α-KDEL. Myc-WDR44 tubules and patches colocalized with Calnexin, an ER transmembrane protein, and α-KDEL, which recognizes luminal ER proteins containing the ER retention signal KDEL. (G) Confocal images of shWDR44a-expressing HeLa cells transfected with GFP-WDR44 ΔFFAT and stained with α-Rab8. GFP–WDR44 ΔFFAT was associated with Rab8-positive tubules. (H) Western blot analysis of an equal protein amount of shRNA-expressing HeLa cell lysates showing specific knockdown of VAPA in shVAPA- and of VAPB in shVAPB-transfected cells. Calnexin was used as loading control. The two parts of the blots were from the same membrane and correspond to identical exposure times. The membrane was initially probed with α-WDR44 and α-VAPB, followed by α-MICAL1 and α-VAPA, and finally α-Calnexin and α-Rab8. (I and J) Confocal images of transfected HeLa cells. GRAF1b/2–RFP tubules colocalized with GFP-VAPA/B (I) but rarely with Calnexin-GFP (J). (K) Confocal images of transfected HeLa cells stained with α-myc and α-Calnexin. When WDR44 was coexpressed, GRAF1b tubules colocalized with an endogenous ER marker. (B, D–G, and I–K) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.