The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks

Area of Science:

• Cell Biology
• Neuroscience
• Biochemistry

Background:

• Endosomal sorting complexes required for transport (ESCRT)-III proteins remodel membranes.
• ESCRT-III complexes, particularly CHMP2B, are hypothesized to form diffusion barriers at post-synaptic spine necks.

Purpose of the Study:

• To investigate the role of CHMP2B in forming diffusion barriers at membrane structures.
• To develop a novel method for reconstituting ESCRT-III proteins in a physiologically relevant membrane topology.

Main Methods:

• Developed a novel assay using giant unilamellar vesicles (GUVs) to reconstitute ESCRT-III proteins.
• Pulled membrane nanotubes from GUVs to mimic post-synaptic spine geometry.
• Investigated CHMP2B binding and polymerization using lipid and protein diffusion assays.

Main Results:

• CHMP2B preferentially binds to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2].
• CHMP2B accumulates at the neck of membrane nanotubes.
• CHMP2B, particularly CHMP2B-ΔC, prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the nanotube neck.

Conclusions:

• CHMP2B polymers formed at membrane necks can function as diffusion barriers.
• This suggests a critical role for CHMP2B in maintaining synaptic spine structure and function.
• The novel GUV-based assay provides a powerful tool for studying ESCRT-III membrane remodeling.