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Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy
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Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

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

Emanuele Cocucci1, Raphaël Gaudin2, Tom Kirchhausen3

  • 1Department 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.

Molecular Biology of the Cell
|September 19, 2014
PubMed

Insights

Dynamin, a GTPase crucial for endocytosis, forms dimers that assemble into collars around membrane necks. This study quantifies dynamin recruitment during vesicle formation, revealing the minimum number required for cell membrane scission.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biophysics

Background:

  • Dynamin is essential for clathrin-mediated endocytosis, a key cellular process for membrane trafficking.
  • Dynamin functions as a GTPase and is recruited to clathrin-coated pits in distinct phases.

Purpose of the Study:

  • To investigate the assembly unit and stoichiometry of dynamin recruitment during endocytosis.
  • To determine the precise number of dynamin molecules required for membrane scission and vesicle release.

Main Methods:

  • Utilized gene-edited cells expressing dynamin2-EGFP for live-cell imaging.
  • Employed single-molecule sensitive TIRF and spinning-disk confocal microscopy.
  • Calibrated imaging with single-molecule EGFP detection to quantify dynamin numbers.

Main Results:

  • Dynamin dimers were identified as the preferred assembly unit at coated pits.
  • Budding coated pits recruit 26–40 dynamins, forming 1–1.5 helical turns.
  • As few as 26 dynamins are sufficient for coated vesicle release in partially depleted cells.

Conclusions:

  • The findings provide quantitative insights into dynamin collar formation and its role in membrane scission.
  • Results constrain mechanistic models of dynamin-mediated membrane fission during endocytosis.

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