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Related Experiment Videos

Understanding living clathrin-coated pits.

Joshua Z Rappoport1, Sandford M Simon, Alexandre Benmerah

  • 1The Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, Box 304, New York, New York 10021, USA.

Traffic (Copenhagen, Denmark)
|April 17, 2004
PubMed
Summary

Studying clathrin-mediated endocytosis in living cells using total internal reflection fluorescence microscopy (TIR-FM) reveals the dynamic nature of clathrin-coated pits and vesicles (CCPs). This advanced imaging challenges existing models of endocytosis.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biophysics

Background:

  • Traditional studies of clathrin-mediated endocytosis relied on biochemical fractionation and in vitro assays.
  • Recent advances allow the study of endocytosis within living cells, offering dynamic insights.
  • Clathrin-mediated endocytosis is a fundamental cellular process for internalizing molecules.

Purpose of the Study:

  • To investigate the dynamic nature of clathrin-coated pits and vesicles (CCPs) in living cells.
  • To visualize and track CCPs and associated proteins directly at the plasma membrane.
  • To challenge and refine existing models of endocytosis using live-cell imaging.

Main Methods:

  • Utilized total internal reflection fluorescence microscopy (TIR-FM), also known as evanescent field microscopy.

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  • Enabled direct observation of cellular events near the adherent plasma membrane.
  • Tracked individual CCPs and CCP-associated proteins like clathrin, dynamin, and AP-2.

    • Main Results:

    • Directly observed the dynamic behavior of CCPs and CCVs in real-time within living cells.
    • Visualized CCP-associated proteins, including clathrin, dynamin, and AP-2, at the site of endocytosis.
    • Observations confirmed some aspects of current models while revealing new complexities.

    Conclusions:

    • Live-cell imaging with TIR-FM provides unprecedented insights into the dynamics of clathrin-mediated endocytosis.
    • Direct observation of key proteins challenges established endocytic models and prompts new research questions.
    • TIR-FM is a powerful technique for studying endocytic processes at the plasma membrane.