Gone with the wind: Watching chaperones disassemble clathrin coats

The protein coat surrounding endocytic clathrin-coated vesicles is rapidly disassembled, in a process catalyzed by the Hsc70 molecular chaperone, immediately after the vesicles are internalized into the cell. A new fluorescence imaging approach has enabled the first detailed, real-time visualization of the uncoating reaction, revealing the unexpected accumulation of multiple Hsc70 molecules on the clathrin coat, as a requisite for coat destabilization by a fluctuation-capture mechanism.

HFSP Cross Disciplinary Fellow Till Böcking and colleagues
authored on Tue, 22 February 2011



When watching a field of dandelions from afar, it is easy to observe a plume of seeds as the wind blows through the field. But how quickly an individual seed head disassembles remains undetermined because seed release is not synchronized for the dandelions in the field. To understand this process in detail, we need to zoom in and record seed release for each single dandelion. This approach allows us to observe different intermediates of seed head disassembly and determine, for example, when dispersal starts and when it is complete. Using a similar  “single-particle” imaging approach, we studied the disassembly of individual clathrin coats, macromolecular structures of about 100-200 nm, five orders of magnitude smaller in size than dandelions. 

Clathrin is the main structural component of the machinery that mediates cargo entrapment and endocytosis of a number of nutrients, hormones, lipoproteins and viruses into eukaryotic cells. Assembly of the three-legged clathrin triskelion into an array of hexagons and pentagons on the intracellular side of the plasma membrane drives invagination of the plasma membrane, which after pinching into the cell interior becomes a clathrin-coated vesicle. The clathrin coat is then rapidly disassembled to allow the naked vesicle to fuse with its endosomal target. This “uncoating” reaction is catalyzed by Hsc70 or heat shock cognate protein 70, an ATP-dependent chaperone that is recruited to the clathrin coat by its co-chaperone auxilin.

Hsc70 is a simple ATP-driven molecular motor: In the ATP-loaded state, Hsc70 is in its open conformation allowing it to associate with its substrate (in this case clathrin). The ensuing ATP hydrolysis results in a closed conformation and tight substrate binding. Nucleotide exchange releases the substrate and resets the cycle by reopening Hsc70. The uncoating activity of Hsc70 in conjunction with auxilin has long been known from ensemble experiments.  To examine in detail how such a simple molecular clamp can mediate the disassembly of a complex macromolecular structure, required the new, single-particle approaches now feasible.

We unraveled the disassemblase mechanism of Hsc70 by using fluorescence microscopy to observe how Hsc70 binding to the clathrin coat leads to its disassembly. We used recombinant proteins to reconstitute fluorescent clathrin coats, captured them onto the modified glass bottom of a microfluidic flow cell and introduced Hsc70 labeled with a different fluorophore. Each clathrin coat appeared as a bright spot in the fluorescence image with an intensity proportional to the number of clathrin trimers. Fluorescence imaging revealed an increase in the Hsc70 fluorescent signal on each individual coat before the simultaneous disappearance of its clathrin and Hsc70 signals as uncoating ensued. Single-particle analysis of several hundred uncoating traces followed at different Hsc70 concentrations, in conjunction with a kinetic model for the uncoating reaction, revealed the following behaviour: Multiple Hsc70 molecules must first bind to the coat so that disassembly can occur only after a threshold level is reached. Once initiated, the disassembly process is a rapid all-or-none reaction that requires the continued presence of Hsc70. Our recent electron cryo-microscopy structure of a stabilized clathrin coat with bound auxilin and Hsc70 shows that a single Hsc70 binds underneath a triskelion and that the combined association of auxilin and Hsc70 to the coat results in a global distortion of its structure. We combined the information obtained by using real-time visualization with the molecular snapshots to propose a molecular mechanism for the uncoating reaction whereby conformational fluctuations throughout the clathrin coat are trapped by Hsc70 molecules long enough that they reinforce each other until a point is reached where the entire lattice is sufficiently destabilized to fall apart. This type of fluctuation-capture mechanism could represent an example of a more general process of chaperone-driven disassembly of macromolecular complexes.  The single-particle imaging approach developed here can be exploited as a powerful tool to study other cellular assembly and disassembly reactions.


Figure: Left: Selected snapshots from a time series (top) showing the fluorescence from clathrin and Hsc70 in a coat visualized using the single-particle uncoating assay (shown schematically at the bottom). Right: The plot shows intensity traces of the clathrin (blue) and Hsc70 (orange) signals of a single coat during the uncoating reaction.


Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Till Böcking, François Aguet, Stephen C Harrison & Tomas Kirchhausen. Nature Structural and Molecular Biology, doi:10.1038/nsmb.1985, published online 30 January 2011.

Other references

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly. Yi Xing, Till Böcking, Matthias Wolf, Nikolaus Grigorieff, Tomas Kirchhausen & Stephen C Harrison. EMBO Journal 2010, 29(3), 655-665.

Pubmed link