FIGURE 4.

Relationship between VPS4 and CHMP2B protrusions. HeLa cells were co-transfected with the indicated plasmids and observed 36 h later. A, in cells transfected with the control plasmid together with GFP-VPS4, VPS4 is distributed homogeneously in the cytoplasm. In cells expressing both GFP-VPS4B and CHMP2B, VPS4 is mainly present in CHMP2B tubes. In contrast, AMSH is not recruited in CHMP2B protrusions made by cells expressing both GFP-AMSH and CHMP2B. B, CHMP2B^L207D/L210D-FLAG, mutated in the MIM domain, accumulates in plasma membrane patches but does not induce protrusions. CHMP2B^intron5, which lacks the C-terminal α6 helix containing the MIM domain, forms everting tubes. GFP-VPS4 is recruited neither in CHMP2B^L207D/L210D-FLAG patches nor in CHMP2B^intron5-FLAG containing tubes. C, the catalytically inactive form of VPS4B (GFP-VPS4B^E235Q) increases the proportion of cells displaying CHMP2B protrusions: HeLa cells were transfected with CHMP2B together with GFP-VPS4B^E235Q and observed 24 h later. For each condition, 200 cells from three independent experiments were counted. Standard error bars are shown. Mann-Whitney statistical analysis was used (p < 0.0011). Scale bars, 20 μm. **, p < 0.0011.