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Related Concept Videos

Pinching-off of Coated Vesicles01:32

Pinching-off of Coated Vesicles

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Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming...
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Clathrin-coated vesicles use endocytosis to transport receptors and lysosomal hydrolases from the Golgi to the lysosome in the late secretory pathway. Clathrin-mediated endocytosis was the first described endocytic process, and Clathrin-coated vesicles remain one of the most well-studied transport vesicles. The molecular machinery that generates clathrin-coated vesicles comprises over 50 proteins that precisely coordinate vesicle formation. Cell surface receptors concentrated in indented sites...
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Membrane-enclosed structures called vesicles transport proteins and lipids across the cell. The vesicles derive their cargo from the plasma membrane, Golgi, ER, or endosome. Coated vesicles are spherical, protein-coated carriers with a 50–100 nm diameter that mediate bidirectional transport between the ER and the Golgi. The distribution of proteins between the ER and Golgi complex is dynamic and is maintained by different coated vesicles. Their formation is driven by the assembly of...
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Related Experiment Video

Updated: May 30, 2025

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy
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SuperResNET - single-molecule network analysis detects changes to clathrin structure induced by small-molecule

Timothy H Wong1, Ismail M Khater2,3, Christian Hallgrimson2

  • 1Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

Journal of Cell Science
|January 27, 2025
PubMed
Summary
This summary is machine-generated.

SuperResNET analysis reveals how endocytosis inhibitors pitstop 2 and dynasore affect clathrin-coated pit morphology. Pitstop 2 arrests clathrin pit formation at early stages, distinct from actin depolymerization effects.

Keywords:
ClathrinDynasoreMachine learningPitstopSmall moleculeSuper resolution

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In vivo and in vitro Studies of Adaptor-clathrin Interaction
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Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
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Area of Science:

  • Cell biology
  • Biophysics
  • Microscopy

Background:

  • Clathrin-mediated endocytosis is crucial for cellular processes.
  • Understanding the precise mechanisms of endocytosis inhibitors is vital.
  • Single-molecule localization microscopy (SMLM) provides high-resolution insights into molecular dynamics.

Purpose of the Study:

  • To apply the SuperResNET pipeline to analyze SMLM dSTORM data.
  • To investigate the effects of pitstop 2, dynasore, and latrunculin A on clathrin-coated pit morphology.
  • To characterize distinct stages of clathrin-coated pit formation and maturation.

Main Methods:

  • Network analysis of point cloud data using SuperResNET.
  • Application of SMLM direct stochastic optical reconstruction microscopy (dSTORM).
  • Analysis of clathrin structures in HeLa and Cos7 cells treated with inhibitors.

Main Results:

  • Identified three classes of clathrin structures: oligomers, pits/vesicles, and clusters.
  • Pitstop 2 and dynasore induced distinct class II structures, suggesting different endocytosis arrest points.
  • Latrunculin A induced large, heterogeneous structures, indicating effects independent of actin depolymerization.
  • Ternary analysis revealed pitstop 2-treated cells had planar clathrin profiles, resembling early pits.

Conclusions:

  • SuperResNET effectively analyzes SMLM data to reveal inhibitor effects on clathrin structures.
  • Pitstop 2 arrests clathrin pit maturation at early stages of formation.
  • This approach allows in situ detection of small molecule effects on cellular targets from SMLM datasets.