title: Dynactin 3D structure: implications for assembly and dynein binding.
abstract: The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High-resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either ≈1MDa assembly. In the present study, we used negative-stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional (3D) structure of native vertebrate dynactin. The 35-nm-long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other end, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in two-dimensional images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7 Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.
authors.affiliation: ERATO Actin Filament Dynamics Project, Japan Science and Technology Agency, c/o RIKEN, Sayo, Hyogo 679-5148, Japan; Laboratory for Structural Biochemistry, RIKEN Harima Institute SPring-8 Center, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Imai
authors.affiliation: ERATO Actin Filament Dynamics Project, Japan Science and Technology Agency, c/o RIKEN, Sayo, Hyogo 679-5148, Japan; Laboratory for Structural Biochemistry, RIKEN Harima Institute SPring-8 Center, Sayo, Hyogo 679-5148, Japan; Structural Biology Research Center and Division of Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Nagoya 464-8601, Japan; JST PRESTO, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Narita
authors.affiliation: ERATO Actin Filament Dynamics Project, Japan Science and Technology Agency, c/o RIKEN, Sayo, Hyogo 679-5148, Japan; Laboratory for Structural Biochemistry, RIKEN Harima Institute SPring-8 Center, Sayo, Hyogo 679-5148, Japan; Structural Biology Research Center and Division of Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Nagoya 464-8601, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Maéda
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA. Electronic address: schroer@jhu.edu.
authors.forename: Trina A
authors.initials: TA
authors.lastname: Schroer
mesh_terms: D051344:Actin Capping Proteins / Q000378:metabolism; D008841:Actin Cytoskeleton; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002642:Chick Embryo; D000072159:Dynactin Complex; D004398:Dyneins / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008840:Microfilament Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008958:Models, Molecular; D011485:Protein Binding
chemical_list: D051344:Actin Capping Proteins; D000072159:Dynactin Complex; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D004398:Dyneins
keywords: 3D EM; actin; dynein; microtubule; single particle analysis