title: Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.
abstract: Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Grassart
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Cheng
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Sun Hae
authors.initials: SH
authors.lastname: Hong
authors.affiliation: Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
authors.forename: Nathan
authors.initials: N
authors.lastname: Zenzer
authors.affiliation: Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
authors.forename: David M
authors.initials: DM
authors.lastname: Briner
authors.affiliation: Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Davis
authors.affiliation: Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Malkov
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 drubin@berkeley.edu.
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
mesh_terms: D000199:Actins / Q000378:metabolism*; D002460:Cell Line; D002469:Cell Separation; D002966:Clathrin / Q000737:chemistry*; D003599:Cytoskeleton / Q000378:metabolism; D034285:Dynamin II / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry; D016678:Genome; D006801:Humans; D007091:Image Processing, Computer-Assisted; D020014:K562 Cells; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D014168:Transferrin
chemical_list: D000199:Actins; D002966:Clathrin; D014168:Transferrin; D034285:Dynamin II