To construct a single-molecule detection system for ESCRTⅢ, an important proteins involved in the development of lip and palate.

The structure of the ESCRTⅢ protein was analyzed, and the amino acids which predicted to have no affection on the structural stability and filament formation were selected for mutation. The mutant proteins were extracted and the stabilities were detected. The efficiency of fluorescence labeling of mutant protein was detected by absorption spectrum. The ability of the mutant protein to form filaments was observed by negative staining electron microscope, and the ability of filament formed by mutant protein to be depolymerized by Vps4 was observed by negative staining electron microscope and analyzed by centrifugal precipitation analysis.

Five sites on ESCRTⅢ protein Snf7, K57, G58, N59, G120 and D122, were selected to mutate to cysteine. K57C mutant protein was unstable. G58C mutant protein had low fluorescence labeling efficiency. Mutation on N59 and G120 mutant protein affected the formation of filament. Only the D122C mutant protein was stable and the labeling efficiency was high. The labeled protein formed filament that could be depolymerized by Vps4 protein.

The Snf7 D122C mutant was screened out, which is suitable for further detection of the depolymerization dynamic process of ESCRTⅢ filaments by the single-molecule technique, so as to provide supports for further study of the effect of ESCRT system on development of palate at the cellular level and in vivo.

Cleft lip and palate; Snf7; ESCRTⅢ; Single-molecule assay; Vps4