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.
Highlights
In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage
giant unilamellar vesicles (GUVs) labelled with 1% of tetra-methyl rhodamine (TMR)-PIP2 were incubated with 0.5 mM purified AP180 and 0.4 mM purified clathrin containing 20 mol% of Alexa Fluor 488-labelled clathrin (AF488Clathrin)
As clathrin/AP180 forms highly curved cages in vitro in the absence of membrane[15], we hypothesized that the combined effect of tension and bending rigidity was responsible for the impairment of full budding by opposing the clathrin polymerization energy
Summary
In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. Clathrin forms flat hexagonal lattices at the plasma membrane of cells[12], and spherical coated vesicles of various sizes, ranging from 35 to 200 nm, in various organisms[13] This variability in size and shape has called into question the initially proposed membrane scaffolding mechanism based on clathrin polymerization. AP180 has an ANTH domain that binds phosphatidylinositol 4,5-bisphosphate (PIP2)[5] It forms shallow pits when reconstituted on lipid monolayers[5] and does not bud the membrane of large unilamellar vesicles (LUVs)[6]. These results suggested that clathrin polymerization energy was not the dominant factor in controlling the shape of clathrin coats. We questioned how parameters of membrane elasticity (that are, tension and rigidity) could affect clathrin polymerization
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