An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors1,2,3,4. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone5,6,7. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.
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We thank R. Schäfer for project support. We acknowledge discussions with J. Morin, Ü. Coskun, A. Honigmann, S. Sturm and T. Leonard, and F. Jülicher, E. Schäfer and K. Simons for reading the manuscript. We thank M. Brandstetter and the electron microscopy facility of the Vienna Biocenter. We thank the Light Microscopy, Protein Expression, Chromatography, and High-throughput Technology Development Studio of the Max Planck Institute of Molecular Cell Biology and Genetics. During part of the work, M.J. was supported by a PhD scholarship of the Böhringer Ingelheim Fonds. M.J.A. was supported by the La Caixa and Deutscher Akademischer Austauschdienst scholarship. R.P. was supported by the National Health and Medical Research Council of Australia (program grant APP1037320 and Senior Principal Research Fellowship 569452) and the Australian Research Council Centre of Excellence (CE140100036). We acknowledge the Australian Microscopy & Microanalysis Research Facility at the Center for Microscopy and Microanalysis at The University of Queensland. S.W.G. was supported by the Deutsche Forschungsgemeinschaft (SPP 1782, GSC 97, GR 3271/2, GR 3271/3, GR 3271/4), the European Research Council (grant number 281903) and the Human Frontier Science Program (RGP0023/2014). This research was supported by the Max Planck Society and funds of the Deutsche Forschungsgemeinschaft (Transregio 83).
The authors declare no competing financial interests.
Reviewer Information Nature thanks C. Schmidt, J. Zimmerberg and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 EEA1 is a predicted extended coiled-coil dimer that binds Rab5 in a GTP-dependent manner and extends outwards from endosomes
a, Human EEA1 in COILS prediction reveals a clear coiled-structure flanked by the Rab5-binding Zn2+-finger on the N terminus and PI(3)P binding FYVE domain on the C terminus. b, Coomassie-stained gel of human EEA1 expressed as a GST fusion in SF+ insect cells and purified by GS affinity, cleaved on resin, and subsequently concentrated and separated from smaller contaminants by size-exclusion chromatography on a Superose 6 column. c, Static light scattering in line with size-exclusion chromatography reveals a molecular mass of 323 kDa, compared with a theoretical molecular mass of 326 kDa for a dimeric protein. d, Purified protein binds Rab5 in both standard and optical tweezer conditions (35% glycerol) in a GTP-dependent manner. GST or GST-Rab5 was purified and conjugated to GS resin, and subsequently nucleotide was exchanged to either GTP-γS or GDP using EDTA-Mg2+-mediated exchange and subsequent wash. The GST resin was then incubated with EEA1 in either the standard or optical tweezers buffer, washed three times, and beads were then blotted for EEA1. e, Recombinant EEA1 binds specifically to PI(3)P liposomes. When mixed with POPC:POPS 85:15 liposomes, no EEA1 is observed in the liposome pellet (CTRL). In contrast, EEA1 is pelleted with control POPC:POPS:PI(3)P 80:15:5 liposomes (PI3P). f, The N-terminal Zn2+-finger and C-terminal FYVE domain of EEA1 were differentially labelled with specific antibodies and STORM microscopy performed to define their localization in HeLa cells. Representative STORM images of EEA1 radial extension from endosome of n = 22. Scale bar, 500 nm. g, h, Primary antibody binding controls for N and C termini. Primary antibodies for the N (g) and C (h) termini were left out of the staining, resulting in no unspecific secondary staining for each. Representative of n = 5. Scale bar, 500 nm.
Extended Data Figure 2 Validation of bead-supported lipid bilayers for optical tweezers, and bead tethering experiment controls and methods.
To optimize the conditions for forming supported lipid bilayers on the 2–10 μm beads, we systematically investigated the dependence of membrane formation on salt and liposome concentration. a, Fluorescent profiles of supported lipid bilayer bead cross sections. At high liposome concentration (100 μM, solid line) during formation of the bilayer on the silica bead, the bead-supported membrane fluorescence intensity is circumferentially homogenous. At lower lipid concentrations (10 and 1 μM, dashed and dotted lines), less than full coverage is achieved and the supported bilayer is inhomogeneous. b, Consistent with previous reports, increasing salt concentrations result in more homogenous membrane coverage. c, Representative examples of the ‘spilled-out’ membrane of beads prepared at 100 mM (top, blue) and 250 mM (bottom, red) NaCl salt and 100 μm liposomes, of n = 5. d, Histogram of the size of membrane spilled from the beads onto the substrate when prepared at 100 and 250 mM NaCl (blue and red, respectively). This indicated that the lower salt samples (blue) were homogenously covered with membrane and that they had little excess present, and therefore the optimal conditions for formation of membrane on the silica beads used in tethering and in optical tweezer experiments. e, Segmentation of beads and vesicles by the SQUASH method. Bead-supported bilayers and vesicles (green and magenta, respectively) were segmented as illustrated by red outlines to determine their co-localization. Representative of n = 1 generated for schematic. f, Methodology comparison for co-localization in GDP and GTP-γS conditions. All methods give P < 0.01 in a two-tailed Student’s t-test. Co-localization by signal is better than by size or object, as vesicles become undercounted at high concentrations. Mean ± s.d., n = 5. g, Co-localization of liposomes (PI(3)P, magenta) to the bead-supported membrane (GFP–Rab5, green) was strictly dependent on GTP-γS. Box–whisker plot with minimum/maximum error, n = 5. h, The co-localization of liposomes to the supported membrane was dependent on EEA1 concentration. At higher concentrations of EEA1, co-localization approached 100%. These concentrations are within the range of the concentration of endogenous protein23. Mean ± s.d., n = 5. i, Time-lapse micrographs of the bead-supported bilayer labelled with GFP–Rab5 (green), and a dynamically tethered vesicle (magenta). Vesicles were observed to tether and reversibly leave the membrane, as well as diffuse about its surface. Images displayed were acquired at 350 ms intervals as z-stacks. Representative of n = 1 to acquire video. Scale bar, 2 μm. j, Example fits for radial line-profile data.
a, COILS prediction for extended EEA1 mutant, revealing removal of most of the discontinuities in the coiled-coil. b, c, The swapped EEA1 mutant has a rearranged coiled-coil. The coiled-coil was split as indicated by red triangles in the original EEA1-WT (b), and the two regions a (shaded green) and b (shaded magenta) were rearranged in a synthetic gene, producing the swapped EEA1 variant maintaining the features and sequence of the original coiled-coil, but in an alternative location (c). d, Full sequence alignment for human EEA1 and the extended and swapped mutants used in the study. The crystal structure (Protein Data Bank accession number 3MJH) for the Zn2+-finger domain is marked in dark blue close to the N terminus. Segment a of the coiled-coil region is marked in green, and segment b in magenta. The crystal structure (Protein Data Bank accession number 1JOC) of the C-terminal FYVE domain and portion of the coiled-coil is marked in cyan. Details of the mutant constructs are found in the Methods.
Extended Data Figure 4 Extended and swapped EEA1 mutants exhibit limited changes in the presence of Rab5:GTP-γS.
a, e, Rotary-shadowed EEA1-extended particles and EEA1-swapped mutants were skeletonized and analysed in ImageJ for contour length (top), resulting in normally distributed contour length histograms. The end-to-end length histograms (bottom) are similarly distributed. These data were collected on N-terminally MBP-tagged samples. Compare with wild-type in Fig. 2b, d; n = 212 for the extended and n = 93 for the swapped variants. b–d, f, g, The EEA1 mutants revealed limited changes to their curvature in the presence of Rab5:GTP-γS (b, f; compare Fig. 2i, j), and therefore minor changes to their contour and end-to-end length histograms (c, g) and radial distribution plots (d, h); n = 80 for the extended and n = 47 for the swapped variants. i, j, Rotary-shadowing electron microscopy of EEA1 in the presence of Rab5:GDP (n = 90), N-terminally MBP-tagged, revealed no change in appearance compared with the absence of Rab5 entirely (Fig. 2a), and no effect of N-terminal tagging relative to wild-type EEA1. k, Radial distribution function of EEA1 in the presence of Rab5:GDP (compare d, h; Fig. 2g); n = 90.
Extended Data Figure 5 Representative segmentation, smoothing and signed curvature measures for EEA1, and averages for EEA1 and mutants.
EEA1 and EEA1 mutants were skeletonized and smoothed using a moving average filter with a window of 8.2 nm, segmented to 300 equally spaced segments and aligned N terminus to C terminus by recognition of an N-terminal MBP-tag. Their curvature was calculated at 15 nm distances along the length of the proteins and plotted. a–c, Representative examples of rotary shadowing derived EEA1 curves. The original data appear in the first panel, with the second panel revealing the data after smoothing for comparison (Methods). The curvature measure, determined by how the tangents to the contour change at a distance of 15 nm along the contour is plotted below. Note that the choice of sign for the curvature measure is arbitrary for each molecule. d, e, Curvature measure and variance of this measure for EEA1 in the presence of Rab5:GDP (green) and EEA1 in the presence of Rab5:GTP-γS (magenta); n = 90, n = 145, respectively. Alignment of EEA1 curvature from the electron microscopy data reveals an increase in curvature over the length of the molecule upon Rab5 binding, whereas the extended and swapped EEA1 variants show no change. All curvature values were taken to be positive given that the N-terminal MBP could be recognized but the handedness of the molecule adsorbed to the grid could not be inferred. Bootstrapping with resampling at full population size was performed for 1,000 iterations to determine errors. f, g, Extended EEA1 variant in the absence (green) and in the presence of Rab5:GTP-γS (magenta); n = 212, n = 80, respectively. h, i, Swapped EEA1 variant in the absence (green) and in the presence of Rab5:GTP-γS (magenta); n = 93, n = 47, respectively.
Extended Data Figure 6 Detailed persistence length and equilibration analysis for EEA1 and variants.
To validate the methodology used for analysis of the persistence lengths, and to assure internal consistency in analysis methods, we systematically applied the analysis to EEA1 (and mutants, see Supplementary Data Table). The skeletonized curves were segmented to 300 equally spaced segments, where θ describes the angle between segments. The tangent–tangent correlations were then determined for the entire ensembles. a–h, To determine the molecular equilibration of EEA1 and variants from 3D to 2D, the kurtosis of the theta distribution (top) was calculated. Full equilibration to 2D gives a value of 3.0, and for 3D the expected value is 1.8 as the angle distributions become Gaussian. As expected, the measured kurtosis is approximately 3.0 until lengths above the persistence length of the molecule, where the equilibration begins to fail. The value at which the kurtosis began to diverge from 2D was taken as the limit for subsequent measurements, as beyond this limit (red shaded region) 3D fluctuations are not retained and as such the consequences of surface adsorption are uncertain. Next, the tangent–tangent correlation was calculated across the ensemble and fitted up to the divergence of the kurtosis (red shaded region).
a, Change-point analysis was used to identify changes in the mean and variance of the combined force signal. An example plot of averaged force (linear combination of signals from both traps) with respect to time. Data have been collected at 1 kHz. Two long transient interactions can be clearly identified. b, c, Cross-correlation of the force signals from each trap are not sufficient to reveal stepwise interactions as they are time-averaged. By applying cross-correlation over a correlation window of 0.8 s (b) or 0.3 s (c), long transient interactions (that is, at ~4 s) could be identified. However, an unbiased identification of short transients (that is, at ~9 s) by this method was not possible. All identified long transient interactions showed characteristic changes in the cross-correlation: anti-correlation as beads are pulled together, and correlation after tethering was established. d, Change-point analysis was used to detect both changes in mean and variance of the combined force signal, and thereby identify transient interactions (red line). This procedure has the additional advantage of defining clear boundaries to stepwise processes. e, The possibility of multiple tethers taking part in the reaction was observed. Averaged force trace for wild-type EEA1 occasionally showed signals consistent with multiple interactions (cyan), in addition to single transient interactions (red). f, Zoom into time series around the transient interaction identified in the previous panel. To a first approximation, the dynamic interactions were fitted as piecewise constant steps (red). Note also two very short (<10 ms) spikes of similar magnitude (to the left and right of identified interaction) occurred but are not used in further analysis. Only transients with a duration longer than 100 ms were analysed. g, To illustrate the sensitivity of the optical tweezer experiments, a noise analysis was performed on the segment outlined in the top panel (yellow, labelled Allan analysis). The Allan deviation (square root of Allan variance, in piconewtons) gives a threshold for detecting a signal change over different averaging windows. All detected transients (blue) are at minimum an order of magnitude above this threshold. To provide perspective, the transient in the above example is indicated as a red dot. h, The entropic collapse force is balanced in the tweezer experiments below its peak value. The balance between the average restoring force in the optical traps (brown) and the entropic collapse force of EEA1 (blue) in the bound state gives the measured equilibrium force and extension (red dot). The schematic assumes the measured capture distance of 195 nm, a persistence length in the Rab5:GTP-bound state of λb = 26 nm, and a contour length of 222 nm. The overall trap response of the dual-trap system is treated as two springs in series with the mean trap stiffness in trap 1 (κ1 = 0.035 ± 0.007 pN/nm) and the mean trap stiffness in trap 2 (κ2 = 0.029 ± 0.007 pN/nm), leading to an overall trap stiffness of κT = 0.0159 pN/nm (brown line). Given these parameters, the predicted equilibrium force in the optical trap for Rab5-bound EEA1 is ~0.6 pN and the predicted equilibrium extension ~160 nm. i, Force changes upon capture for Rab5:GTP-bound EEA1 and the extended and swapped variants. Force was measured from change-point analysis for transient interactions between EEA1 beads and Rab5:GTP beads. To test binding per se, the force change for 10×His-EEA1 beads tethered to Ni-NTA beads was similarly determined from established connections. For 10×His-EEA1, no transient interactions could be observed. Median change in force and 95% confidence interval from bootstrapping with resampling (lower and upper bounds at (2.5%, 97.5%)) were determined. EEA1, 0.37 (0.31, 0.46) pN; extended, 0.39 (0.35, 0.42) pN; swapped, 0.45 (0.41, 0.56) pN; 10×His, 0.19 (0.14, 0.22) pN. j, Capture distances defined at the proximal distance upon which transient interactions were observed for Rab5-bound EEA1 and the extended and swapped variants. Median capture distance and 95% confidence interval from bootstrapping with resampling (lower and upper bounds at (2.5%, 97.5%)) were determined. EEA1, 168 (141, 182) nm; extended, 195 (189, 199) nm; swapped, 183 (179, 189) nm; 10×His, 157 (120, 196) nm; n = 60, 93, 27, 24 per condition respectively. k, Mechanical work is performed as the tether collapses. The mechanical work performed during the relaxation to the new equilibrium extension is the integral under the force–extension curve. The exact value of the extracted work depends both on the capture distance (the extension at the moment of persistence length change) and on the release distance (the extension at the moment when Rab5 unbinds). The uncertainties in these extensions are different for the two positions, reflecting the different longitudinal fluctuations of the rigid or the flexible tether (λflexible = 26 nm (blue arrows), λrigid = 300 nm (magenta arrows)). For example, for a relaxation between the capture distance, dcapture ≈ 195 nm and the release extension, drelease ≈ 122 nm, the extracted mechanical work is W ≈ 14 kBT.
Extended Data Figure 8 EEA1 mutants incapable of undergoing entropic collapse result in defects in endosomal trafficking.
a, b, Automated confocal immunofluorescence images (n = 30 each) of HeLa EEA1-KO and standard HeLa cells. EEA1 (green) and Rab5 (magenta). Scale bar, 10 μm. c, Western blot of HeLa and HeLa EEA1-KO clonal cell line for EEA1 and Rab5. d, e, g, h, Automated confocal images (n = 30 each) of HeLa EEA1-KO cells expressing no EEA1 (KO, d), rescued with wild-type EEA1 (rescue, e) or extended and swapped mutants (g, h). Cells were pulsed with fluorescently labelled cargo (LDL) (green) for 10 min, fixed and immunostained for Rab5 (magenta) and EEA1 (for EEA1, see Fig. 4). Magnified insets of endosomes are depicted at arrows. Scale bar, 10 μm. f, Relative complexity of Rab5 endosomes per cell. Each Rab5 endosome is segmented, and the segmented object requires a defined number of 2D Gaussian functions, hereby referred to as complexity. Relative to wild type, HeLa EEA1-KOs (black line) had a significantly reduced number of endosomes of high complexity (>3.0), but more endosomes defined simply by one or two Gaussian functions. Rescue experiments (red) revealed no significant difference in complexity. In contrast, both extended and swapped mutants (blue and green respectively) had significantly fewer simple endosomes of low complexity, and significantly more of higher complexity. Mean ± s.d., n = 30. i, Histogram of fluorescence intensity of EEA1 per cell. KO cell lines had a sharp peak of intensity at background levels, whereas wild-type HeLa cells had a normal distribution. Grey box represents threshold levels of EEA1 intensity per cell taken for analysis. j–l, EGF uptake experiments. Confocal images of HeLa EEA1-KOs expressing wild-type EEA1 (rescue, j) or extended and swapped mutants (g, h). Cells were pulsed with fluorescently labelled EGF (green) for 10 min, fixed and immunostained for EEA1 (magenta). Images shown are maximum intensity projections. Scale bar, 5 μm. m, HeLa EEA1-KO cells in which the swapped EEA1 mutant was reintroduced showed clusters of vesicles and more rarely the classical endosomal morphology. The clusters were clearly delineated by a zone of cytoplasm with a distinct density. Representative of n = 19. Scale bars, 2 μm. n, Further quantifications, and the swapped mutant ultrastructural phenotype. Fraction of endosomal surface containing filamentous material for HeLa and HeLa EEA1-KOs. Box–whisker plot with minimum/maximum values, n = 22, 24 endosomes. **P < 0.01, two-tailed Student’s t-test. o, Distance measured between endosome and tethered vesicles (HeLa) or between vesicles within large clusters (extended) (surface-to-surface, n = 158 and 623 for HeLa and extended respectively; ***P < 10−4, two-tailed Student’s t-test).
EEA1 in the absence of Rab5 is predominantly extended. The initial five segments of the curves from rotary shadowing electron microscopy were aligned and the curves plotted with the end position highlighted (dots). Grey concentric hemispheres demarcate 50, 100, 150 and 200 nm extensions from the origin. The end positions therefore resulted in a cloud of empirical positions for the EEA1 N terminus of EEA1 (left), and reveal the overall change in conformational space that can be occupied by EEA1 when bound to Rab5:GTP-γS (right). b, Bouquet plots for the extended EEA1 variant. c, Bouquet plots for the swapped EEA1 variant.
This file contains the uncropped blot images, a Supplementary Discussion, Supplementary References and Supplementary Table 1. (PDF 694 kb)
Representative midplane images taken from z-stacks of a spinning-disc confocal image sequence of 10 μm bead-supported bilayers imaged with GFP-Rab5 at 35 ms per plane. Vesicles were imaged by RhoDPPE with simultaneous excitation. EEA1 was present at an intermediate concentration of 100 nM in order to visualize the reversible nature of the reconstitution (AVI 1321 kb)
Reconstructed z-stack of the bead-based tethering assay, reconstructed z-stack illustrates homogeneity of tethered vesicle distribution, density, and demonstrates three-dimensional nature of vesicle diffusion. Stacks were assembled in Fiji. Conditions are identical to Video 1. 10 μm bead-supported bilayers imaged with GFP-Rab5 at 35 ms per plane. Vesicles were imaged by RhoDPPE with simultaneous excitation. EEA1 was present at an intermediate concentration of 100 nM in order to visualize the reversible nature of the reconstitution. (AVI 1852 kb)
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Murray, D., Jahnel, M., Lauer, J. et al. An endosomal tether undergoes an entropic collapse to bring vesicles together. Nature 537, 107–111 (2016). https://doi.org/10.1038/nature19326
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