Cell Biology

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Science  24 Sep 2010:
Vol. 329, Issue 5999, pp. 1577
DOI: 10.1126/science.329.5999.1577-a
CREDIT: WU ET AL., NAT. CELL BIOL. 12, 902 (2010)

The reconstitution of membrane trafficking events in cell-free systems has contributed to understanding the mechanisms of vesicle targeting to and fusion with membranes. Wu et al. have now reconstituted endocytic budding from plasma membrane sheets. Fluorescently labeled mammalian cell plasma membranes were incubated at 37°C in the presence of cytosol, ATP, and either GTP or its nonhydrolyzable analog GTP-γ-S. In the presence of GTP-γ-S, the plasma membrane invaginated into numerous clathrin-associated tubules as large as 2 µm in length, and the subsequent addition of GTP led to the release of numerous vesicles. A protein known to promote membrane tubulation, FBP17 (red), associated with the GTP-γ-S–generated invaginations, as did clathrin (green) and dynamin. Blocking clathrin assembly blocked the production of membrane invaginations and tubules, and reducing cytosolic FBP-17 blocked the formation of membrane tubules. Further studies in this system should help to clarify the various stages of clathrin-coated pit formation, invagination, and scission.

Nat. Cell Biol. 12, 902 (2010).

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