Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

Abstract

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

Keywords: cargo binding; deafness; microfilaments; myosins.

Figure 1.. Human deafness mutant R1166X affects myosin VI intracellular localisation.

(A) Schematic diagram of myosin VI showing the positions of the human deafness mutant R1166X in the tail domain and the rigor mutant K157R in the motor domain. The large and small inserts (from alternative splicing) and the cargo-binding motifs RRL, WWY and IWE along with their cargo adaptors are also indicated. (B) RPE cells were transfected with WT GFP-tagged LI myosin VI (GFP-MVI-LI) or mutant GFP-MVI-LI (R1166X) and double labelled for GFP and clathrin. In addition, RPE cells were transfected with WT GFP-tagged NI myosin VI (GFP-MVI-NI) or mutant GFP-MVI-NI (R1166X) and double labelled with the early endosomal marker APPL1 or actin. Scale bar, 10 µm.

Figure 2.. R1166X mutation increases the amount of myosin VI in the cytosol pellet fraction.

(A) RPE cells were transfected with WT full-length GFP-tagged myosin VI-NI, myosin VI R1166X and the rigor mutant, K157R and the distribution of myosin VI and loading controls, EGFR and actin, between supernatant (S) and pellet (P) was analysed by an actin pelleting assay followed by quantitative immunoblotting. (B) Quantitative immunoblotting of myosin VI distribution between pellet and supernatant fractions is shown (±SEM, n = 5, ***P < 0.001, *P < 0.05).

Figure 3.. Deletion of amino acids 1246–1265 results in shift of myosin VI from supernatant to pellet fraction.

(A) Schematic diagram showing the positions of myosin VI truncations and mutations. (B and C) RPE cells were transfected with WT or mutant myosin VI with increasing 20 amino acid truncations from the C-terminus and the distribution between pellet and supernatant fractions was analysed as before by quantitative immunoblotting (±SEM, n = 4, **P < 0.01). (D) RPE cells were transfected with GFP-full-length myosin VI-NI or GFP-myosin VI 1–1185 and double labelled with GFP and actin. Scale bar, 10 µm.

Figure 4.. Mutation of W1253 causes redistribution of myosin VI from supernatant to pellet fraction.

(A) Further truncations were made between amino acids 1245 and 1265 and analysed as before by quantitative immunoblotting (±SEM, n = 4, **P < 0.01). (B and C) Full-length myosin VI constructs with point mutations in the region of W1253 were expressed in RPE cells and analysed as before (±SEM, n = 3, ***P < 0.001).

Figure 5.. Binding of myosin VI to cargo adaptor proteins requires amino acid W1253.

The mammalian two-hybrid assay was used to test binding of full-length myosin VI carrying various point mutations to Dab2 (A) or Tom1 (B) (±SEM, n = 5, 3, respectively). (C) Sequence alignment of Dab2, Tom1, Tom1L2 and LMTK2 reveals homologous regions as myosin VI-binding domains. (D) RPE cells were transfected with GFP empty vector, with GFP-Tom1 WT or GFP-Tom1 mutant W423A/L424A followed by GFP immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies to myosin VI, GFP and Tollip. (E) The mammalian two-hybrid assay was used to test the binding of full-length myosin VI with the W1253A mutation to the WWY-binding partners, Dab2, Tom1 and LMTK2, and the RRL-binding partners, GIPC, NDP52 and optineurin (±SEM, n = 4). (F) Pull-down assay of NDP52 (lane 2) or Tom1 (lane 3) or both (lane 4) with GST-myosin VI CBD. After pull-down with glutathione-sepharose beads, the amount of NDP52, Tom1 or both binding to GST-myosin VI CBD was visualised by SDS–PAGE.

Figure 6.. Loss of cargo adaptor-binding sites increases myosin VI in the pellet fraction.

(A and B) RPE cells were transfected with WT or mutant myosin VI with mutations in either the RRL or the WWY adaptor-binding sites and the distribution between pellet and supernatant fractions was analysed by quantitative immunoblotting (±SEM, n = 5, **P < 0.01, *P < 0.05). (C and D) The mammalian two-hybrid assay was used to identify R1108 as the essential amino acid in the RRL motif for myosin VI binding to GIPC (C) or NDP52 (D) (±SEM, n = 3).