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Figures
- Fig. 1.
EHD1 is required for mitochondrial homeostasis. (A–D) RPE cells were either mock treated (A,C) or treated with EHD1 siRNA for 72 h (B,D) and immunostained for the mitochondrial membrane marker Tom20. C and D are images of higher magnitude to visualize mitochondrial elongation. (E) The efficacy of the EHD1-depletion for A–D is demonstrated by immunoblotting lysates from mock and EHD1-depleted RPE cells. (F–H) The Mito Morphology Macro plugin in ImageJ was used to quantify mean±s.d. for mitochondrial size, perimeter and circularity, in three independent experiments each using 10 cells per treatment. *P<0.05 (one-tailed Student's t-test).
- Fig. 2.
Mitochondrial dynamics are impaired upon EHD1 depletion. Live imaging was performed on RPE cells incubated with Mitotracker Red and either mock treated (A) or treated with EHD1 siRNA for 72 h (B). 4-slice z-section images were taken every 15 s for 5 min for each treatment, and compiled to a maximal projection image. The arrows depict examples of fission events in mock-treated cells.
- Fig. 3.
EHD1 plays a regulatory role in mitochondrial fission. (A–D) RPE cells were either mock treated (A,B) or treated with EHD1 siRNA for 72 h (C,D), followed by incubation with STS for the last 1 h of treatment (B,D) or without the drug (A,C). (E–G) The Mito Morphology Macro plugin in ImageJ was used for quantifying mean±s.d. for mitochondrial size, perimeter and circularity in three independent experiments each using 10 cells per treatment. *P<0.05; n.s., not statistically significant (one-tailed Student's t-test).
- Fig. 4.
EHD1 interacts with Mul1. (A) Model for the potential role of EHD1 in regulating mitochondrial dynamics via Mul1. Under normal conditions, the ubiquitin ligase Mul1 is released from an interaction with VPS35 and the retromer components (including EHD1), and relocates to the mitochondrial membrane, where it ubiquitylates Mfn2, inducing its proteasomal degradation and promoting normal mitochondrial fission. Upon EHD1 depletion, Mul1 would be retained in association with VPS35 and the retromer, preventing Mfn2 degradation and thus enhancing mitochondrial membrane fusion. (B) GST pulldown from bovine brain cytosol was performed with GST only, a GST-tagged EH domain of EHD1 (GST–EH1) and GST–EHD1. Eluates were immunoblotted with antibodies against MICAL-L1 (top panel), as a positive interactor with EHD1, and Mul1 (middle panel). GST fusion protein samples were immunoblotted with anti-GST (bottom panel). (C) Co-immunoprecipitation (IP) of proteins from a HeLa cell lysate using anti-Mul1 (αMul1), and immunoblotted with anti-Vps26 and anti-rabankyrin-5 antibodies. 25 kDa immunoglobulin light chains detected by the secondary anti-light chain antibody are indicated in the bottom panel.
- Fig. 5.
Rabankyrin-5 mediates the interaction between EHD1 and Mul1, and its depletion induces an elongated mitochondrial network similar to that observed upon EHD1 depletion. (A,B) RPE cells were either mock treated (A) or treated with rabankyrin-5 siRNA for 72 h (B) and immunostained for the mitochondrial membrane marker Tom20. (C) The Mito Morphology Macro plugin in ImageJ was used for quantifying mean±s.d. for mitochondrial size, perimeter and circularity in three independent experiments each using 10 cells per treatment. *P<0.05 (one-tailed Student's t-test). (D) HeLa cells were either mock treated or treated with rabankyrin-5 siRNA, lysed, and subjected to a GST–EHD1 pulldown, and immunoblotted with Mul1 (upper panel). The efficacy of the rabankyrin-5-depletion is demonstrated by immunoblotting lysates from mock-treated and rabankyrin-5-depleted cells (bottom two panels).
- Fig. 6.
Depletion of EHD1, rabankyrin-5 or VPS35 does not induce Mfn2 accumulation. HeLa cells were either mock treated, or treated with EHD1, rabankyrin-5 or Vps35 siRNA for 72 h. Depletion efficacy was validated by immunoblotting with antibodies against EHD1, rabankyrin-5 and VPS35 (A; top three panels), and the effect of the siRNA was assessed with antibodies against Mfn2 (A; second panel from the top), Drp1 (A; third panel from the top) and actin (A; bottom panel). (B–G) Densitometric quantification from three separate experiments. *P<0.05 (one-tailed Student's t-test).
- Fig. 7.
Depletion of EHD1 and rabankyrin-5 results in reduced and sequestered VPS35, respectively. (A–D) HeLa cells were either mock treated, treated with EHD1 siRNA (A) or treated with rabankyrin-5 (Rank-5) siRNA (C) for 72 h and immunoblotted for VPS35, EHD1, Rank-5 and actin. The asterisk (in A) indicates reduced VPS35 protein levels. (B,D) Quantification of protein levels from three independent experiments. *P<0.05 (one-tailed Student's t-test). (E–J) RPE cells were either mock treated (E,G,I) or treated with rabankyrin-5 siRNA for 72 h (F,H,J), and immunostained for VPS35 and the Golgi membrane marker GM130. Regions of interest were drawn with a dashed line around the GM130 Golgi stain (I,J) and superimposed in the merged images (E,F) and in the VPS35-stained images (G,H; note, the region of interest is not shown in G and H so the VPS35 distribution pattern can be observed more clearly). Scale bar: 10 μm. (K) ImageJ was used to quantify the mean±s.d. for fluorescence of VPS35 localized to the central Golgi region marked by the regions of interest in three independent experiments each using 10 cells per treatment. *P<0.05 (one-tailed Student's t-test). (L) Current working model showing the proposed mechanism for EHD1 regulation of mitochondrial dynamics. In this scenario, EHD1 might act in facilitating fission of vesicles that transport VPS35 from endosomes to the mitochondrial membrane. VPS35 might then interact with inactive Drp1, removing it from the mitochondrial membrane and facilitating the function of active Drp1 leading to mitochondrial fission. Thus, the absence of EHD1 might prevent this transport step and lead to elongated mitochondria.
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