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PMID: 25232009 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.

Molecular biology of the cell ·Vol. 25 ·No. 22 ·2014-11-05 ·Pages 3595-609

Cocucci E, Gaudin R, Kirchhausen T

Abstract

Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.

MeSH Terms
Animals Base Sequence Cattle Cell Line, Tumor Clathrin/metabolism Clathrin-Coated Vesicles/metabolism,ultrastructure Coated Pits, Cell-Membrane/metabolism,ultrastructure Dynamin II Dynamins/antagonists & inhibitors,chemistry,genetics,metabolism Endocytosis Gene Expression Regulation Genes, Reporter Green Fluorescent Proteins/genetics,metabolism Humans Microscopy, Fluorescence Models, Molecular Molecular Sequence Data Protein Multimerization Protein Transport RNA, Small Interfering/genetics,metabolism Signal Transduction
Chemicals
Clathrin RNA, Small Interfering enhanced green fluorescent protein Green Fluorescent Proteins DNM2 protein, human Dynamin II Dynamins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Cocucci Emanuele
Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115.
Gaudin Raphaël
Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115.
Kirchhausen Tom
Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115 kirchhausen@crystal.harvard.edu.
References (59)
59 references, click to expand
  1. Dynasore, a cell-permeable inhibitor of dynamin.
    Dev Cell. 2006 Jun;10(6):839-50 PMID: 16740485
  2. Membrane fission: model for intermediate structures.
    Biophys J. 2003 Jul;85(1):85-96 PMID: 12829467
  3. Dual role of BAR domain-containing proteins in regulating vesicle release catalyzed by the GTPase, dynamin-2.
    J Biol Chem. 2013 Aug 30;288(35):25119-25128 PMID: 23861397
  4. Structures and mechanisms of vesicle coat components and multisubunit tethering complexes.
    Curr Opin Cell Biol. 2012 Aug;24(4):475-83 PMID: 22728063
  5. Visualizing density maps with UCSF Chimera.
    J Struct Biol. 2007 Jan;157(1):281-7 PMID: 16963278
  6. Robust single-particle tracking in live-cell time-lapse sequences.
    Nat Methods. 2008 Aug;5(8):695-702 PMID: 18641657
  7. Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating.
    Nat Struct Mol Biol. 2011 Mar;18(3):295-301 PMID: 21278753
  8. Mechanics of dynamin-mediated membrane fission.
    Annu Rev Biophys. 2013;42:629-49 PMID: 23541160
  9. Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.
    Nat Cell Biol. 2011 Mar;13(3):331-7 PMID: 21297641
  10. A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.
    PLoS Biol. 2012;10(4):e1001302 PMID: 22505844
  11. A quantitative method for the detection of edges in noisy time-series.
    Philos Trans R Soc Lond B Biol Sci. 1998 Dec 29;353(1378):1969-81 PMID: 10098215
  12. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.
    Nat Cell Biol. 2002 Sep;4(9):691-8 PMID: 12198492
  13. Molecular mechanism and physiological functions of clathrin-mediated endocytosis.
    Nat Rev Mol Cell Biol. 2011 Jul 22;12(8):517-33 PMID: 21779028
  14. Linking data to models: data regression.
    Nat Rev Mol Cell Biol. 2006 Nov;7(11):813-9 PMID: 17006434
  15. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells.
    Cell. 2005 May 20;121(4):593-606 PMID: 15907472
  16. Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction.
    Cell. 2012 Oct 26;151(3):619-29 PMID: 23101629
  17. A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke.
    Cell. 2011 Sep 30;147(1):209-22 PMID: 21962517
  18. Crystal structure of nucleotide-free dynamin.
    Nature. 2011 Sep 18;477(7366):556-60 PMID: 21927000
  19. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.
    J Cell Biol. 1994 Nov;127(4):915-34 PMID: 7962076
  20. Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring.
    Nat Cell Biol. 1999 May;1(1):27-32 PMID: 10559860
  21. The first five seconds in the life of a clathrin-coated pit.
    Cell. 2012 Aug 3;150(3):495-507 PMID: 22863004
  22. Molecular cytogenetic analysis of 11 new breast cancer cell lines.
    Br J Cancer. 1999 Dec;81(8):1328-34 PMID: 10604729
  23. Dynamin-catalyzed membrane fission requires coordinated GTP hydrolysis.
    PLoS One. 2013;8(1):e55691 PMID: 23383266
  24. A transcription activator-like effector toolbox for genome engineering.
    Nat Protoc. 2012 Jan 05;7(1):171-92 PMID: 22222791
  25. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.
    J Cell Biol. 1989 Feb;108(2):389-400 PMID: 2563728
  26. A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.
    PLoS Biol. 2011 Mar;9(3):e1000604 PMID: 21445324
  27. The stalk region of dynamin drives the constriction of dynamin tubes.
    Nat Struct Mol Biol. 2004 Jun;11(6):574-5 PMID: 15133500
  28. Dynamics of intracellular clathrin/AP1- and clathrin/AP3-containing carriers.
    Cell Rep. 2012 Nov 29;2(5):1111-9 PMID: 23103167
  29. Activation of dynamin GTPase is a result of positive cooperativity.
    J Biol Chem. 1994 Dec 9;269(49):30842-7 PMID: 7983015
  30. An intramolecular signaling element that modulates dynamin function in vitro and in vivo.
    Mol Biol Cell. 2009 Aug;20(15):3561-71 PMID: 19515832
  31. The relationship between the number of synaptic vesicles and the amount of transmitter released.
    J Neurosci. 1989 Jun;9(6):1937-42 PMID: 2566663
  32. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission.
    Nature. 2006 May 25;441(7092):528-31 PMID: 16648839
  33. Dynamin self-assembly stimulates its GTPase activity.
    J Biol Chem. 1996 Sep 13;271(37):22310-4 PMID: 8798389
  34. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis.
    Nat Cell Biol. 2011 Aug 14;13(9):1124-31 PMID: 21841790
  35. A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis.
    Science. 2007 Apr 27;316(5824):570-4 PMID: 17463283
  36. The crystal structure of dynamin.
    Nature. 2011 Sep 18;477(7366):561-6 PMID: 21927001
  37. Endocytosis by random initiation and stabilization of clathrin-coated pits.
    Cell. 2004 Sep 3;118(5):591-605 PMID: 15339664
  38. Correlation between self-association modes and GTPase activation of dynamin.
    J Protein Chem. 1999 Apr;18(3):277-90 PMID: 10395446
  39. Geometric catalysis of membrane fission driven by flexible dynamin rings.
    Science. 2013 Mar 22;339(6126):1433-6 PMID: 23520112
  40. Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells.
    Mol Biol Cell. 2008 Dec;19(12):5347-59 PMID: 18923138
  41. Domain structure and intramolecular regulation of dynamin GTPase.
    EMBO J. 1997 Nov 17;16(22):6676-83 PMID: 9362482
  42. The dynamin superfamily: universal membrane tubulation and fission molecules?
    Nat Rev Mol Cell Biol. 2004 Feb;5(2):133-47 PMID: 15040446
  43. Molecular structures of coat and coat-associated proteins: function follows form.
    Curr Opin Cell Biol. 2006 Aug;18(4):395-406 PMID: 16806884
  44. Molecular structure, function, and dynamics of clathrin-mediated membrane traffic.
    Cold Spring Harb Perspect Biol. 2014 May 01;6(5):a016725 PMID: 24789820
  45. The dynamins: redundant or distinct functions for an expanding family of related GTPases?
    Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):377-84 PMID: 9012790
  46. Three-dimensional reconstruction of dynamin in the constricted state.
    Nat Cell Biol. 2001 Oct;3(10):922-6 PMID: 11584275
  47. Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.
    J Cell Biol. 2014 Jun 9;205(5):721-35 PMID: 24891602
  48. Dynamin undergoes a GTP-dependent conformational change causing vesiculation.
    Cell. 1998 Jun 12;93(6):1021-9 PMID: 9635431
  49. Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.
    Dev Cell. 2013 Aug 12;26(3):279-91 PMID: 23891661
  50. GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission.
    Cell. 2008 Dec 26;135(7):1276-86 PMID: 19084269
  51. Molecular basis for recognition of dilysine trafficking motifs by COPI.
    Dev Cell. 2012 Dec 11;23(6):1255-62 PMID: 23177648
  52. Membrane insertion of the pleckstrin homology domain variable loop 1 is critical for dynamin-catalyzed vesicle scission.
    Mol Biol Cell. 2009 Nov;20(22):4630-9 PMID: 19776347
  53. Three dynamin-encoding genes are differentially expressed in developing rat brain.
    J Neurochem. 1996 Sep;67(3):927-31 PMID: 8752097
  54. A model for dynamin self-assembly based on binding between three different protein domains.
    J Biol Chem. 1999 May 21;274(21):14942-7 PMID: 10329695
  55. Dynamin forms polymeric complexes in the presence of lipid vesicles. Characterization of chemically cross-linked dynamin molecules.
    J Biol Chem. 1995 Nov 3;270(44):26707-14 PMID: 7592898
  56. Building a fission machine--structural insights into dynamin assembly and activation.
    J Cell Sci. 2013 Jul 1;126(Pt 13):2773-84 PMID: 23781021
  57. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding.
    Nature. 1995 Mar 9;374(6518):190-2 PMID: 7877694
  58. Sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin AP-2 complexes.
    EMBO J. 1996 Nov 1;15(21):5789-95 PMID: 8918456
  59. G domain dimerization controls dynamin's assembly-stimulated GTPase activity.
    Nature. 2010 May 27;465(7297):435-40 PMID: 20428113
Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1939-4586
DOI
10.1091/mbc.E14-07-1240
Email
kirchhausen@crystal.harvard.edu
Published
2014-11-05
Epub
2014-00-17
Pages
3595-609
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC4230619
Subset
IM
Grants
NIGMS NIH HHS · R01 GM075252 · United States
NIAID NIH HHS · U54 AI057159 · United States
NIGMS NIH HHS · GM-075252 · United States
External Links
PubMed source DOI: 10.1091/mbc.E14-07-1240 PMC Full Text View journal details
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