title: Filopodia formation by crosslinking of F-actin with fascin in two different binding manners.
abstract: Filopodia are finger-like protrusions at the leading edge of migrating cells that play a crucial antennal function during cell motility. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by virtue of fascin, which plays a central role in actin filament bundling. However, the molecular mechanisms underlying their formation remain unclear. Here, we observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three-dimensional structure by cryo-electron tomography and image processing; the actin filament bundling structure by fascin was clarified at high resolution under physiological conditions. It was found that actin filaments in vivo were more numerous than in bundles reconstructed in vitro, and each filopodial actin filament had limited variability in helical twisting. In addition, statistical analysis of actin filament bundles unveiled their detailed architecture. In filopodia, actin filaments had highly ordered structures, and the shift between cross-links of each adjacent actin filament was approximately 2.7 nm, similar to the monomer repeat of actin filaments. We then proposed a plausible actin-fascin cross-link model at the amino acid level and identified three fascin binding sites on two adjacent actin filaments: one filament bound fascin at two discrete, widely separated regions and the other bound fascin in a single small region. We propose that these two different binding modalities should confer rigid bundles that retain flexibility and dynamic performance. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka, Japan.
authors.forename: Shinji
authors.identifier: http://orcid.org/0000-0001-7372-5529
authors.initials: S
authors.lastname: Aramaki
authors.affiliation: Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, Japan.
authors.forename: Kouta
authors.initials: K
authors.lastname: Mayanagi
authors.affiliation: Graduate School of Faculty of Medicine, Osaka City University, 1-4-3 Asahi-Machi, Abeno-Ku, Osaka, Japan.
authors.forename: Mingyue
authors.initials: M
authors.lastname: Jin
authors.affiliation: Application Laboratory, FEI Company Japan Ltd, 2-13-34 Konan, Minato-Ku, Tokyo, Japan.|Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, 7-1, Mihogaoka, Ibaraki, Osaka, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoyama
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka, Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Yasunaga
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D020285:Cryoelectron Microscopy; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats
chemical_list: D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; C020324:fascin
keywords: 3D reconstruction; cell biology; cryo electron tomography; image processing; structural biology