Clathrin exchange during clathrin-mediated endocytosis.

Area of Science:

• Cell biology
• Molecular and cellular dynamics
• Membrane trafficking

Background:

• Clathrin-mediated endocytosis involves the invagination of clathrin-coated pits into vesicles.
• Structural rearrangement of clathrin is necessary as planar pits become spherical vesicles.
• The mechanism of clathrin structural rearrangement, specifically clathrin exchange, remains unclear.

Purpose of the Study:

• To investigate clathrin exchange dynamics in clathrin-coated pits and vesicles.
• To determine whether clathrin exchange is a prerequisite for structural rearrangement during endocytosis.
• To differentiate clathrin exchange mechanisms in pits versus baskets.

Main Methods:

• In vitro assays using clathrin-coated vesicles and baskets with Hsc70 and ATP.
• Fluorescence recovery after photobleaching (FRAP) studies in HeLa cells expressing GFP-clathrin light chains.
• Inhibition of endocytosis using dynamin mutants or cholesterol depletion.
• Chemical treatments (potassium depletion, hypertonic sucrose) to induce clathrin cage formation.

Main Results:

• In vitro, clathrin in coated vesicles and baskets showed no exchange with free clathrin, even with Hsc70 and ATP.
• In vivo, FRAP revealed rapid, ATP-dependent exchange of clathrin in coated pits with the cytosol, independent of endocytosis progression.
• Potassium depletion and hypertonic sucrose blocked both endocytosis and clathrin exchange in coated pits, forming stable cages.

Conclusions:

• ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, not clathrin baskets.
• This exchange likely facilitates the structural rearrangement of clathrin during pit invagination.
• Clathrin exchange dynamics differ significantly between clathrin-coated pits and pre-formed clathrin structures like baskets.