Fig 2: EHD2 oligomers mediate a link of caveolae to F-actin. (A) Confocal images of primary human fibroblasts immunostained with EHD2 and CAV1 antibodies and stained with fluorescent Phalloidin to visualize filamentous actin. Cells were transfected with control siRNA or EHD2 siRNA. (B) Confocal images of human fibroblasts transfected with control siRNA or EHD2 siRNA and treated for 5 min with 5 µM CytoD. CAV1 and Phalloidin signal is depicted. Side panels show enlargements of indicated regions in the merged images. Arrowheads highlight CAV1 close to actin patches. (C) HeLa cells stably expressing CAV1–EGFP were treated with control siRNA or EHD2 siRNA and transfected with RFP–actin (not depicted). Image depicts the CAV1–EGFP maximum intensity projection of all frames of a 7.5-min TIR-FM movie (90 frames, frame rate 0.2 Hz, penetration depth 110 nm) starting upon addition of 5 µM CytoD. Distinct trajectories of caveolae moving in the PM plane due to actin retraction can only be seen in control conditions. (D) CAV1-/- cells were transfected with EHD2-I157Q–EGFP and RFP–actin (not depicted), treated with CytoD and live imaged as in C. (D) Maximum intensity projection of EHD2-I157Q–EGFP signal reveals retracting puncta. (E) Confocal images of CAV1-/- cells, transfected with EHD2-I157Q–EGFP, treated for 5 min with 5 µM CytoD and stained with fluorescent Phalloidin. Side panels show enlargements of indicated region in the merged image. Arrowheads highlight EHD2 close to actin patches. (F) Confocal images of CAV1-/- cells, transfected with EHD2-T94A–EGFP, treated and analysed as in E. All scale bars 10 µm.

Fig 3: The ATPase domain regulates EHD2 dynamics and function. (A) Representative confocal images of FRAP experiments with EHD2-I157Q–EGFP expressing cells. No fluorescence recovery at initially bleached spots can be detected during the recorded time. (B) FRAP curves of EHD2–EGFP or EHD2-I157Q–EGFP-transfected CV1 cells (as in Figure 4D). Data points present mean values±s.e.m., n=10 or 8 for EHD2–EGFP or EHD2-I157Q–EGFP. (C) Representative confocal image of a CV1 cell co-expressing EHD2-I157Q–EGFP and EHD2–mCherry for 12 h. Cells were fixed and stained for endogenous CAV1. Arrows in the magnification highlight EHD2-I157Q-positive spots that have weak/no EHD2 wild-type staining and colocalize with CAV1. (D) CV1 cells transfected with EHD2–EGFP and CAV1–mCherry were incubated for 40 min in ATP-depletion media and analysed by confocal microscopy. Magnification highlights colocalization of EHD2 and CAV1. (E) Confocal image of MEF CAV1-/- cell transfected with EHD2-I157Q–EGFP for 6 h. (F) Relative CAV1 fluorescence 12 min after bleaching peripheral regions in HeLa cells, stably expressing CAV1–EGFP and transfected with mCherry control (n=11) or EHD2-T72A–mCherry (n=11) plasmids. Significance of mean differences between the conditions was calculated with a two-tailed unpaired t-test. ***P-value<0.0001. (G) Representative confocal image of a CV1 cell co-expressing EHD2–EGFP and EHD2-T72A–mCherry. Cells were fixed and stained for endogenous CAV1. Arrows in the magnification highlight that CAV1-positive spots are devoid of wild-type EHD2 and EHD2-T72A. All scale bars 10 µm. (H) A model on the role of oligomer formation and the ATP cycle in EHD2 association with caveolae and dynamic exchange. (1) Newly synthesized EHD2 dimers bind ATP and are subsequently targeted to the plasma membrane via ionic interactions with negatively charged lipids. (2) Upon lipid binding, EHD2 dimers assemble into 60–75S oligomers via interactions of EH domain and KPF/NPF motifs in adjacent EHD2 dimers. (3) Association of EHD2 complexes with caveolae requires ATP hydrolysis, which may be stimulated at caveolae. (4) EHD2 proteins undergo dynamic exchange at caveolae, which requires a new cycle of ATP binding and hydrolysis. EHD2 mediates a link to actin fibres that run in close proximity to caveolae.

Fig 4: Mutations in EHD2 impair association with caveolae and formation of large EHD2 complexes. (A) Domain structure of EHD2 with generated point and deletion mutants and table summarizing effects of these mutations described in previous studies (Guilherme et al, 2004; Daumke et al, 2007). (B) Confocal images of CV1 cells transiently expressing EGFP fusions of EHD2 mutants for 6–8 h. Cells were fixed and stained for endogenous CAV1 (only shown for EHD2-I157Q–EGFP as the other mutants did not colocalize with CAV1). Scale bar 10 µm. (C) Cell lysates prepared from 3T3-L1 cells or HeLa cells expressing EHD2–EGFP were run through 10–40% sucrose velocity gradients and fractions analysed by SDS–PAGE/western blot. In both cell lysates, EHD2 was present in distinct oligomeric species sedimenting at 8S, 60S, and 75S (Hayer et al, 2010a). 3T3-L1 fractions were analysed with anti-EHD2 antibodies and HeLa fractions with anti-GFP antibodies. (D) EHD2 mutants were analysed for their ability to form protein complexes with sedimentation coefficients >60S. EGFP fusions of mutant proteins were expressed for 6–12 h in HeLa cells and lysates run through sucrose velocity gradients (as in C). Fractions were analysed with anti-EHD2 antibodies. Bars present mean±s.e.m. of relative EHD2 signal in fractions 9–12 (‘oligomeric EHD2’) from three independent experiments.

Fig 5: EHD2 downregulation is associated with breast cancer cell aggressiveness. (a–c) Representative transmitted light images (left) and quantification (right) of cell migration using a wound healing assay in Hs578T cells EHD2 depleted (siEHD2) or not (CTRL) (a), in MDA-MB-231 cells overexpressing EHD2 or not (CTRL) (b), and in MDA-MB-436 cells overexpressing EHD2 or not (CTRL) (c). Scale bar =10 µm. Cell migration into the wound site was assessed after 16 h. (d–f) Quantification of invasion using Transwell chamber inserts in Hs578T cells EHD2 depleted (siEHD2) or not (CTRL) in the absence (negative control) or presence of serum (d), in MDA-MB-231 cells overexpressing EHD2 or not (CTRL) (e), and in MDA-MB-436 cells overexpressing EHD2 or not (CTRL) (f). (g) Measurement of cell proliferation of the Hs578T cells EHD2 depleted (siEHD2) or not (CTRL), and in MDA-MB-436 cells overexpressing EHD2 or not (CTRL). (h,i) Quantification of IL-8 mRNA levels in Hs578T cells EHD2 depleted (siEHD2) or not (CTRL) (h), and in MDA-MB-231 cells overexpressing EHD2 (i); n = 3 independent experiments; ns = non-significant; *P < 0.05; **P < 0.01; (a–c,e–i) two tailed t-test; (d) Bonferroni’s multiple comparison test; mean ± s.e.m.