A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats - PubMed (original) (raw)

A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats

Mohammed Saleem et al. Nat Commun. 2015.

Abstract

In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. However, the impact of membrane elastic parameters on the assembly and shape of clathrin lattices has never been experimentally evaluated. Here, we show that membrane tension opposes clathrin polymerization. We reconstitute clathrin budding in vitro with giant unilamellar vesicles (GUVs), purified adaptors and clathrin. By changing the osmotic conditions, we find that clathrin coats cause extensive budding of GUVs under low membrane tension while polymerizing into shallow pits under moderate tension. High tension fully inhibits polymerization. Theoretically, we predict the tension values for which transitions between different clathrin coat shapes occur. We measure the changes in membrane tension during clathrin polymerization, and use our theoretical framework to estimate the polymerization energy from these data. Our results show that membrane tension controls clathrin-mediated budding by varying the membrane budding energy.

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Figures

Figure 1

Figure 1. Reconstitution of clathrin polymerization on GUVs and effects of osmotic shocks.

(a) GUVs labelled with 1 mol% TMR-PIP2 (red) incubated with AF488-clathrin alone (green) for 5 min. (b) Same conditions as in a but with AP180/AF488-clathrin. (c) Fluorescence recovery after photobleaching experiments of the AF488-clathrin coat on the membrane (see text). Scale bars, 5 μm (a–c). (d,e) TEM micrographs of GUVs coated with clathrin (scale bars, 200 nm (d) and 100 nm (e)). (f) GUVs labelled with TMR-PIP2 (red) were incubated with AP180/AF488-clathrin in three osmotic conditions: hypotonic (hypo), isotonic (iso) and hypertonic (hyper). Scale bar, 5 μm. (g) Mean fluorescence intensity of the clathrin binding (see Methods) for each osmotic condition, with s.e. (***P<0.0001 in _t_-test). (h) Statistics of vesicle appearance (large, small or no deformation) for the three osmotic conditions. (i) GUVs labelled with TMR-PIP2 (red) were incubated with AF488-AP180 under various osmotic conditions—hypotonic (hypo), isotonic (iso) and hypertonic (hyper). Scale bar, 5 μm. For all the fluorescence experiments, _n_=30–45 under each condition from at least three independent experiments.

Figure 2

Figure 2. TEM images of GUVs incubated with AP180/clathrin under different osmotic conditions.

(a) Hypotonic conditions. Scale bars, 1 μm (top row) and 200 nm (bottom row). (b) Isotonic conditions. Scale bar, 200 nm. (c) Hypertonic conditions. Scale bars, 1 μm (top row) and 200 nm (bottom row). (d) Statistics of GUVs appearance observed by TEM under different conditions. (e) Micrographs of clathrin lattices in isotonic conditions (scale bar, 200 nm) with a high-magnification image (scale bar, 100 nm (bottom)) and (f) micrographs of clathrin lattices in hypertonic conditions showing tabulation of the membrane (scale bar, 200 nm) with a high-magnification image (inset, scale bar, 100 nm). For electron microscopy experiments, _n_=372 (hypotonic), _n_=54 (isotonic) and _n_=134 (hypertonic) from at least three independent experiments.

Figure 3

Figure 3. Model and clathrin polymerization energy measurements.

(a) The four possible states of a single bud considered in the model: bare membrane (no clathrin binding), shallow partial budding, deep partial budding and full budding (yielding a closed bud). Parameterization of the bud is shown on the deep partial bud as discussed in the text. (b) Our mathematical model predicts the state of a membrane densely packed with contiguous buds. Our fitting of the model parameters (see main text) predicts a bud depth ≃2 nm. (c) Phase diagram predicted by our model, showing the predicted budding state as a function of the scaled tension and polymerization energy (logarithmic plot). (d) Experimental set-up for the measurement of the clathrin polymerization energy: OT, optical tweezers; GUV, giant unilamellar vesicle. (e) Confocal images of AF488-clathrin and TMR-PIP2 channel (right images) before and after clathrin injection (inverted contrast). Scale bar, 10 μm. (f) Plot showing tube force versus time during addition of clathrin. (g) Three-dimensional phase diagram with experimental data from e and f (linear plot). The clathrin polymerization energy was measured from 15 independent experiments.

Figure 4

Figure 4. Clathrin coat rupture experiments.

(a) Schematic and confocal images of AP180/AF488-clathrin-coated GUVs before and after being aspirated using a pipette. Arrows show the clathrin-uncoated part of the membrane tongue. (b) Length of the tongue versus aspiration pressure (Δ_P_). (c) AF488-clathrin before and after aspiration (tongue fully released). Arrowheads indicate cracks in the clathrin coat. Scale bars, 5 μm. The experiment was repeated 12 times to measure the average aspiration pressure Δ_P_ for coat rupture.

Figure 5

Figure 5. Effect of high bending rigidity and epsin on the clathrin budding abilities.

(a) AF488-clathrin/AP180 does not bind to GUVs with high bending rigidity (see Methods) under isotonic conditions. (b) AF488-clathrin does not bind to high bending rigidity GUVs in hypo-, iso- and hypertonic conditions. (c) Number of vesicles showing AP180/clathrin binding or no binding under various external buffer conditions. (d) A high binding rigidity did not preclude binding of AP180. (e) AF488-clathrin binding and membrane deformation in the presence of unlabelled epsin were observed under varying osmotic conditions, except in the hypotonic conditions where a fraction of the vesicles remained uncoated. (f) Percentage of vesicles with no, small or large deformation after AF488-clathrin/epsin binding. Scale bar, 5 μm. For all the fluorescence experiments, _n_=30–45 under each condition from at least three independent experiments.

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