Fig. 3.

A conserved W-acidic motif in nesprin-2 localizes KLC to the nuclear envelope. (A) Schematic of nesprin-2 Giant and nesprin-4 highlighting the previously mapped KLC-binding domains in each molecule (Roux et al., 2009; Schneider et al., 2011). (B) Alignment of nesprin sequences. The conserved LEWD motifs in nesprin-2, nesprin-1 and nesprin-4 are highlighted; numbering for nesprin-1 and nesprin-2 is based on the Giant isoform sequences; frog sequences are currently incomplete and have been left unnumbered; top panel indicates degree of conservation. (C) Schematic of the EGFP-tagged nesprin-2 fragments used in the following co-immunoprecipitation assays. (D) COS7 cells were transfected with HA-KLC2 and either wild-type EGFP-nesprin-2 fragments or EGFP-nesprin-2 fragments harboring WD/AA mutations in the LEWD motif. Immunoprecipitation of EGFP or EGFP-nesprin-2 constructs, followed by immunoblot analysis indicates that binding of KLC2 is abolished by mutations in the LEWD motif. (E) Co-immunoprecipitation experiments were performed using HA-KLC2 constructs with point mutations in the binding groove for the W-acidic motif. (F) Co-immunoprecipitation of HA-KLC2 R251D, N287L and R312E with EGFP-nesprin-2 (6146-6799) is significantly reduced (mean±s.e.m.; from n=3 independent experiments; ANOVA, *P<0.05, **P<0.01). (G,H) Myotubes were treated with siRNA to KLC1 and KLC2, and transfected with HA-KLC2 wild-type or mutant constructs. (G) Myotubes were stained for HA-KLC2. Scale bar: 20 µm. (H) As depicted, quantification of fluorescence intensity along a 100-pixel line centered at the NE was performed for the HA-KLC2 signal in these treated myotubes. Fluorescence intensity was normalized to the mean of the first 10 pixels of each line to account for variation in HA-KLC2 expression levels between myotubes (mean±s.e.m.; n=10 nuclei per condition from five myotubes). See supplementary material Fig. S3 for additional co-immunoprecipitation experiments.