ESCRT-III family proteins catalyze membrane remodeling processes that stabilize and constrict membrane structures. It was proposed that stable ESCRT-III complexes containing CHMP2B could establish diffusion barriers at post-synaptic spine neck. In order to better understand this process, we developed a novel method based on fusion of Giant Unilamellar Vesicles to reconstitute ESCRT-III proteins inside GUVs, from which membrane nanotubes are pulled. The new assay ensures that ESCRT-III proteins polymerize only when they become exposed to physiologically relevant membrane topology mimicking the complex geometry of post-synaptic spines. We establish that CHMP2B, both as full-length and with a C-terminal deletion (ΔC), preferentially binds to membranes containing PI(4,5)P2. Moreover, we show that CHMP2B preferentially accumulates at the neck of membrane nanotubes, and provide evidence that CHMP2B-ΔC prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the tube neck. This indicates that CHMP2B polymers formed at a membrane neck may function as a diffusion barrier, a potential important function of CHMP2B to maintain synaptic spine structures.