Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography.
Kukulski, W., Schorb, M., Kaksonen, M. & Briggs, J.A.
Cell. 2012 Aug 3;150(3):508-20. doi: 10.1016/j.cell.2012.05.046.
Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are approximately 100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.
Structure of the immature retroviral capsid at 8 A resolution by cryo-electron microscopy.
Bharat, T.A., Davey, N.E., Ulbrich, P., Riches, J.D., de Marco, A., Rumlova, M., Sachse, C., Ruml, T. & Briggs, J.A.
Nature. 2012 Jul 19;487(7407):385-9.
The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid). Assembly of an infectious virion proceeds in two stages. In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation. However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-A resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.
The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions.
Faini, M., Prinz, S., Beck, R., Schorb, M., Riches, J.D., Bacia, K., Brugger, B., Wieland, F.T. & Briggs, J.A.
Science. 2012 Jun 15;336(6087):1451-4. Epub 2012 May 24.
Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.