MICAL-L1 links EHD1 to tubular recycling endosomes and regulates receptor recycling

Abstract

Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.

Figure 1.

MICAL-L1 interacts with EHD1 via the first of its two NPF motifs. (A) Schematic representation of MICAL-L1 domain organization. MICAL-L1 contains an N-terminal calponin homology (CH) domain; a Lin11, Isl-1, Mec-3 (LIM) domain; two NPF motifs; and two C-terminal coiled-coil (CC) domains. (B) The S. cerevisiae yeast strain AH109 was cotransformed with the indicated GAL4-binding domain (GAL4bd) fusion constructs and Galbd-p53 (control), together with the indicated GAL4 transcription activation (GAL4ad) fusion products: Gal4ad-EHD1 or GAL4ad-SV40 large T-antigen (control). Cotransformants were assayed for their growth on nonselective (+HIS) and selective (−HIS) media. (C) Bacterially expressed, purified recombinant GST-EH-domain of EHD1 (top; GST-EH1) or GST-only (middle) was incubated with lysates from HeLa cells transfected with either WT GFP-MICAL-L1 (WT), GFP- MICAL-L1 with both NPF motifs mutated (ΔNPF1+ΔNPF2), GFP-MICAL-L1 with the first NPF motif mutated (ΔNPF1), or GFP-MICAL-L1 with the second NPF motif mutated (ΔNPF2). The bound proteins were resolved by 8% reducing SDS-PAGE, transferred to nitrocellulose, and immunoblotted with mouse anti-GFP, followed by anti-mouse HRP-conjugated antibodies. ECL was used for detection, and 6% of the total input is shown in the bottom panel. (D) HeLa cells were either transfected with Myc-EHD1 alone (lanes 1, 3, and A) or HA-MICAL-L1 alone (lane B) or cotransfected either with HA-MICAL-L1 and Myc-EHD1 (lanes 2 and 4) or GFP and Myc-EHD1 (lanes C–F). After 48 h, cells were lysed and subjected to immunoprecipitations with anti-HA antibody-conjugated agarose beads (lanes 3, 4, and B), with anti-Myc antibodies (lane A), or with anti-GFP antibodies (lanes D and F). Immunoprecipitates and total cell lysates (as indicated) were resolved by 8% nonreducing SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-EHD1, anti-HA, and anti-GFP antibodies followed by detection with anti-rabbit and anti-mouse HRP-conjugated antibodies. ECL was used for detection. Lanes 1′ and 2′ show longer exposures of lanes 1 and 2, and 2% of the total input was loaded in lanes 1, 2, C, and E.

Figure 2.

MICAL-L1 colocalizes with EHD1 on tubular endosomes. (A–C) HeLa cells on coverslips were transiently cotransfected with Myc-EHD1 and HA-MICAL-L1. After 24 h, cells were fixed and incubated with rabbit anti-Myc and mouse anti-HA antibodies. After washing, cells were then incubated with Alexa Fluor 568–conjugated goat anti-rabbit and 488–conjugated goat anti-mouse antibody and mounted on coverslides for confocal microscopy analysis. (D–F) Untransfected HeLa cells on coverslips were fixed and incubated with affinity-purified polyclonal rabbit anti-EHD1 and mouse anti-MICAL-L1 antibodies and detected using Alexa Fluor 488–conjugated goat anti-rabbit and 568–conjugated goat anti-mouse antibody. Arrows (A–C) depict representative tubules containing both proteins. Insets (D–F) depict higher magnification of the boxed areas. Bar, 10 μm.

Figure 3.

MICAL-L1 associates with tubular membranes independently of EHD1. (A–F) HeLa cells on coverslips were transiently transfected with Myc-EHD1 ΔEH (A and B), Myc-EHD1 K483E (C and D), and Myc-EHD1 K483E+W485A (E and F). After 24 h, cells were fixed and incubated with rabbit anti-Myc antibody and mouse anti-MICAL-L1 antibodies and detected using Alexa Fluor 488–conjugated goat anti-rabbit and Alexa Fluor 568–conjugated goat anti-mouse antibody. Dashed areas indicate transfected cells. (G) HeLa cells growing on 35-mm plates were either mock-treated or treated with EHD1-siRNA and harvested after 72 h. Cells were then lysed and proteins separated by 8% SDS-PAGE before immunoblotting with affinity-purified anti-EHD1 antibodies (top) and monoclonal anti-actin antibody to validate equal protein loading (bottom) followed by anti-rabbit and anti-mouse HRP-conjugated antibodies. ECL was used for detection. (H and I) HeLa cells on coverslips were mock-treated (H) or treated with EHD1-siRNA (I). After 72 h of treatment with siRNA, cells were fixed and stained for endogenous MICAL-L1 followed by 488–conjugated goat anti-mouse secondary antibody. Bar, 10 μm. (J) HeLa cells on coverslips were transfected and processed as described in A–F. Approximately 100 untransfected or cells transfected either with Myc-EHD1 ΔEH, Myc-EHD1 K483E, and Myc-EHD1 K483E+W485A were scored as “containing tubules” or “lacking tubules” from three independent experiments. Error bars, SE. (K) Bacterially expressed, purified recombinant GST, GST-EH-domain of EHD1 (GST-EH-1), or GST-EH domain of EHD1K483E (GST-EH-1 K483E) was incubated with lysates from HeLa cells. The bound proteins were resolved by 8% reducing SDS-PAGE, transferred to nitrocellulose, and immunoblotted with mouse anti-MICAL-L1 to detect binding to endogenous EHD1. After incubation with secondary anti-mouse HRP-conjugated antibodies, ECL was then used for detection.

Figure 4.

The C-terminal CC region of MICAL-L1 is essential and sufficient for association with tubular membranes. (A–H) HeLa cells on coverslips were transiently transfected with the GFP-MICAL-L1 CH domain only (residues 1–100, A), GFP-MICAL-L1 CH and LIM domains only (residues 1-213, B), GFP-MICAL-L1 Δ CCs (residues 1-714, C), HA-MICAL-L1 CCs only (residues 672-863, D), HA-MICAL-L1 WT CCs (residues 700-863, E), HA-MICAL-L1 CCs: site 1 mutated to alanine residues (F), HA-MICAL-L1 CCs: site 2 mutated to alanine residues (G), HA-MICAL-L1 CCs: site 1 + site 2 mutated to alanine residues (H). After 24 h, cells were fixed and directly analyzed or incubated with mouse anti-HA antibody followed by the appropriate secondary antibodies. (I) HA-MICAL-L1 WT CCs (residues 700–863) was subjected to further deletion/truncation analysis as depicted, expressed in HeLa cells, and analyzed for localization to tubular membranes. (J) HeLa cells growing on 60 mm plates were transiently transfected with either HA-tagged WT CCs (700–863; CC-only), HA-tagged WT CCs with alanine mutations at site 1 (CC-mutation site 1), HA-tagged WT CCs with alanine mutations at site 2 (CC-mutation site 2) or HA-tagged WT CCs with alanine mutations at both sites 1 and 2 (CC-mutation site 1 + site 2). After 48 h, the cells were homogenized in ice-cold homogenization buffer, and postnuclear supernatants were separated to membrane and cytosol fractions by ultracentrifugation. The samples were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either anti-HA (top), anti-Tf receptor antibodies (control for membrane fraction, middle) or anti-cytochrome C (control for cytosolic fraction, bottom) followed by anti-mouse HRP-conjugated antibodies. ECL was used for detection. Densitometric analysis was performed on three independent experiments to measure percent membrane-bound. The ratios of membrane-associated to the total protein were then calculated and converted to percentages. Error bars, SE; n = 5 for WT CCs and n = 3 for all three mutants. Significance (p < 0.05) was determined by ANOVA. (K–N) HeLa cells on coverslips were mock-treated (K and M) or treated with MICAL-L1-siRNA (L and N). After 48 h, the cells were transfected with siRNA-resistant HA-tagged WT CCs (700–863, siR-HA-CC-only, K and L) or with siRNA-resistant HA-tagged WT CCs with alanine mutations at both sites 1 and 2 (siR-HA-CC-mutation site 1 + 2, M and N). After an additional 24 h, the cells were fixed and incubated with mouse anti-HA antibodies followed by staining with Alexa Fluor 488–conjugated goat anti-mouse antibody. (O) HeLa cells growing on 35-mm plates were either mock-treated or treated with MICAL-L1-siRNA. After 48 h, the cells were transfected with siR-HA-CC-only or with siR-HA-CC-mutation site 1 + 2. After an additional 24 h, the cells were lysed and proteins separated by 8% SDS-PAGE were subjected to immunoblotting with anti-MICAL-L1 antibodies (top) and monoclonal anti-HA antibody to verify expression of the transfected constructs (bottom), followed by anti-mouse HRP-conjugated antibodies. ECL was used for detection. Bar, 10 μm.

Figure 5.

MICAL-L1 is required for the association of EHD1 with tubular membranes. (A–D) HeLa cells on coverslips were mock-treated (A and B) or treated with MICAL-L1-siRNA (C and D). After 48 h, the cells were transfected with Myc-EHD1 (B and D). After an additional 24 h, the cells were fixed and incubated with rabbit anti-Myc antibody and mouse anti-MICAL-L1 antibodies followed by staining with Alexa Fluor 488–conjugated goat anti-rabbit and Alexa Fluor 568–conjugated goat anti-mouse antibody. (E and F) HeLa cells on coverslips were mock-treated (E) or treated with MICAL-L1-siRNA (F). Seventy-two hours later the cells were fixed and stained for endogenous EHD1 (E and F) followed by Alexa Fluor 568–conjugated goat anti-rabbit antibody. (G–I) MICAL-L1-siRNA-treated cells were cotransfected with an siRNA-resistant HA-MICAL-L1 construct and Myc-EHD1. Cells were fixed, permeabilized, and immunostained with anti-HA (G and I) and anti-Myc (H and I) followed by the appropriate secondary antibodies. Bar, 10 μm. (J) HeLa cells growing on 35-mm plates were either mock-treated or treated with MICAL-L1-siRNA and harvested after 72 h. Cells were then lysed, and proteins separated by 8% SDS-PAGE were subjected to immunoblotting with anti-MICAL-L1 antibodies (top) and monoclonal anti-actin antibody to validate equal protein loading (bottom), followed by anti-mouse HRP-conjugated antibodies. ECL was used for detection. (K) HeLa cells on coverslips were either mock-treated or treated with MICAL-L1-siRNA. After 48 h, the cells were either transfected with only Myc-EHD1 (mock and MICAL-L1-siRNA) or cotransfected with both Myc-EHD1 and siRNA-resistant HA-MICAL-L1 (siRNA + Rescue) and stained as described above. Approximately 100 cells transfected with Myc-EHD1 from three independent experiments were counted for each set of treatments. Error bars, SE. Sunbursts denote Myc-EHD1-transfected cells (A–D), stars denote cells transfected with Myc-EHD1, but not HA-MICAL-L1 (H).

Figure 6.

MICAL-L1 is responsible for the colocalization and coassociation of Rab8a and EHD1 on tubular membranes. (A–D) HeLa cells on coverslips were transiently triple-transfected with HA-MICAL-L1 (A and D), Myc-EHD1 (B and D), and Cherry-Rab8a (C and D). After 24 h, cells were fixed and incubated with rabbit anti-Myc antibody and mouse anti-HA antibody. The cells were then incubated with Alexa Fluor 405–conjugated goat anti-rabbit and Alexa Fluor 488–conjugated goat anti-mouse antibody and mounted on coverslides. Bar, 10 μm. (E–J) HeLa cells on coverslips were mock-treated (E–G) or treated with MICAL-L1-siRNA (H–J). After 48 h, the cells were cotransfected with Myc-EHD1 and Cherry-Rab8a. Twenty-four hours later the cells were fixed and incubated with rabbit anti-Myc antibody followed by staining with Alexa Fluor 488–conjugated goat anti-rabbit antibody. (K) Bacterially expressed, recombinant GST or GST fused to the EH domain of EHD1 (GST-EH1) was incubated with lysates from HeLa cells either mock-treated or treated with MICAL-L1-siRNA. The bound proteins were resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with mouse anti-Rab8a (top) followed by anti-mouse HRP-conjugated antibodies. The same blot was stripped with 3 M guanidinium isothiocyanate and probed with mouse anti-MICAL-L1 antibody (middle). ECL was used for detection. GST- and GST-EH1–purified protein bands shown in bottom panel were visualized with Coomassie blue dye staining. (L) HeLa cells growing on 35-mm plates were either mock-treated or treated with Rab8a-siRNA and harvested after 72 h. Cells were then lysed, and proteins were separated by 10% SDS-PAGE and subjected to immunoblotting with anti-Rab8a antibodies (top) and monoclonal anti-actin antibody to validate equal protein loading (bottom) followed by anti-mouse HRP-conjugated antibodies. ECL was used for detection. (M and N) HeLa cells on coverslips were treated with Rab8a-siRNA. After 72 h, the cells were fixed and stained for endogenous MICAL-L1 (M) and endogenous EHD1 (N) followed by the appropriate secondary antibodies. Arrows denote colocalization on tubules. Bars, 10 μm. (O) The S. cerevisiae yeast strain AH109 was cotransformed with the indicated GAL4-binding domain (GAL4bd) fusion constructs and Galbd-p53 (control), together with the indicated GAL4 transcription activation (GAL4ad) fusion constructs and GAL4ad-SV40 large T-antigen (control). Cotransformants were assayed for their growth on nonselective (+HIS) and selective (−HIS) media.

Figure 7.

MICAL-L1 regulates the recycling of Tf receptor and β1 integrins back to the plasma membrane. (A–D) HeLa cells on coverslips were mock-treated (A and B) or treated with MICAL-L1-siRNA (C and D). Seventy-two hours later the cells were serum-starved for 30 min and pulsed with Tf-568 for 10 min and fixed (A and C), or first “chased” in complete medium for 25 min at 37°C before fixation (B and D). (E) Quantitative analysis of the percentage of internalized Tf remaining after 15 min of pulse and 40 min of chase was done. Approximately 80 cells from three independent experiments were counted for each set of treatments. Error bars, SE. (F–I) HeLa cells on coverslips were mock-treated (F and G) or treated with MICAL-L1-siRNA (H and I) for 72 h. Cells were then starved and pulsed with anti-β1 integrin antibody for 1 h and fixed (F and H) or pulsed for 1 h and chased in complete media for 3 additional hours at 37°C before fixation (G and I). (J) Quantitative analysis of the percentage of β1 integrins remaining after 3 h of chase was done as described for Tf. Significance (p < 0.05) was determined by the Student's t test for independent samples. Bar, 10 μm.

Figure 8.

Potential model depicting MICAL-L1 interactions with EHD1 and Rab8a on tubular recycling endosome membranes. MICAL-L1 is recruited to the membranes of tubular recycling endosomes (blue bars) by its C-terminal CC region. Through the binding of its first NPF motif with the EHD1 EH domain (EH1), it recruits and/or stabilizes EHD1 on these tubules. EHD1 is further stabilized on these membranes by the ability of EH1 to directly bind phosphoinositides, specifically PI4P. MICAL-L1 also directly binds to GTP-bound Rab8a and recruits/and or stabilizes it on the tubular membranes; such binding might be simultaneous or independent of EHD1. Localization of EHD1 to the tubular recycling compartment facilitates receptor recycling from the ERC back to the plasma membrane. Rab8a localization to tubular membranes may also play a minor role in EHD1 and MICAL-L1–mediated endocytic recycling pathway. However, as both EHD1 and Rab8a have been implicated in trafficking from the Golgi compartment, their association through MICAL-L1 might be important for trafficking of cargo from Golgi to the plasma membrane or Golgi-to-ERC transport. ERC, endocytic recycling compartment; PI4P, phosphotidylinositol-4-phosphate.