The quick-freeze, deep-etch technique developed in this laboratory will be used to study three key processes in eukaryotes, namely, the formation and dissolution of clathrin cages, the assembly and disassembly of an extracellular matrix, and recognition between gametic cells. Ongoing studies of all three systems are described to demonstrate the unique contributions the quick-freeze, deep-etch technique can make to their understanding. Critical is the ability of the technique to visualize proteins in their native state and after experimental manipulation. Thus, for example, the components of the clathrin cages can be viewed as individual proteins (""""""""triskelions"""""""", """"""""cagin"""""""", etc.) or in various stages of assembly, disassembly, and association with membrane surfaces. Particularly valuable are images of interactions between fibrous proteins, such as those that constitute the extracellular matrix and the sexual agglutinins of Chlamydomonas, since these are impossible to decipher by any other electron microscopic technique. A broad range of experiments is proposed to probe the """"""""rules"""""""" by which these self-assembling sets of proteins carry out their biological functions. Many of these experiments will involve the structural analysis of proteins purified in two collaborating laboratories; in addition, new approaches are described for studying such processes as clathrin/membrane interactions. These studies will hopefully increase our basic understanding of how extracellular proteins interact, how cells regulate their uptake of proteins and their internal membrane traffic, and how cells recognize each other.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Washington University
Schools of Medicine
Saint Louis
United States
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Heuser, John E (2011) The origins and evolution of freeze-etch electron microscopy. J Electron Microsc (Tokyo) 60 Suppl 1:S3-29
Huang, Jing; Roth, Robyn; Heuser, John E et al. (2009) Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress. Blood 113:1589-97
Hanson, Phyllis I; Roth, Robyn; Lin, Yuan et al. (2008) Plasma membrane deformation by circular arrays of ESCRT-III protein filaments. J Cell Biol 180:389-402
Huang, Ren-Huai; Wang, Ying; Roth, Robyn et al. (2008) Assembly of Weibel-Palade body-like tubules from N-terminal domains of von Willebrand factor. Proc Natl Acad Sci U S A 105:482-7
Cardone, Giovanni; Winkler, Dennis C; Trus, Benes L et al. (2007) Visualization of the herpes simplex virus portal in situ by cryo-electron tomography. Virology 361:426-34
McCarren, J; Heuser, J; Roth, R et al. (2005) Inactivation of swmA results in the loss of an outer cell layer in a swimming synechococcus strain. J Bacteriol 187:224-30
Heuser, John (2005) Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic ""honeycomb"" surface coat. J Cell Biol 169:269-83
Szajner, Patricia; Weisberg, Andrea S; Lebowitz, Jacob et al. (2005) External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice. J Cell Biol 170:971-81
Bretschneider, Till; Diez, Stefan; Anderson, Kurt et al. (2004) Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr Biol 14:1-10
Engqvist-Goldstein, Asa E Y; Zhang, Claire X; Carreno, Sebastien et al. (2004) RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery. Mol Biol Cell 15:1666-79

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