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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: J Med Genet. 2018 Mar 23;55(7):479–488. doi: 10.1136/jmedgenet-2017-105221

Figure 4.

Figure 4

Functional evaluation of wild type and mutated forms of human GFP-tagged espin using LLC-PK1-CL4 epithelial cells. Actin-based microvilli are labelled with phalloidin (red). (A–C) Wild type GFP–espin was efficiently targeted to microvilli and caused their elongation. (D–F) GFP–espin with the p.Lys663Thrfs*1 (c.1988delAGAG) human DFNB36 mutation neither targeted microvilli nor caused microvillar elongation and instead was found throughout the cell, including the nucleus. (G–I) GFP–espin with the USH1M (p.790-795delRRDLLR) mutation was found in the cytoplasm, nucleus and the junctional belt at the cell periphery and did not cause microvillar elongation. (J–O) Replacement of the RRDLLR sequence with KKEIIK (J–L) gave results similar to wild type; however, replacement with the neutral amino acids SQSTTS revealed reduced expression of protein and eliminated microvillar targeting and elongation (M–O). Scale bar in panel O is 10 μm and applies to all panels. (P) Quantification of microvillus length present at the surface of LLC-PK1-CL4 cells transfected with various GFP–espin constructs. ****P<0.005.