Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions.
Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.
Egile
Coumaran
C
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
Rouiller
Isabelle
I
Xu
Xiao-Ping
XP
Volkmann
Niels
N
Li
Rong
R
Hanein
Dorit
D
D051377:Actin-Related Protein 2 / Q000737:chemistry*; D051378:Actin-Related Protein 3 / Q000737:chemistry*; D000199:Actins / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry; D003712:Dendrites / Q000378:metabolism; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000737:chemistry; D008840:Microfilament Proteins / Q000737:chemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000737:chemistry; D011817:Rabbits; D012984:Software
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D011721:Pyrenes; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins