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Fig. 1. The C-termini of Nesprins are conserved and sufficient for NE localisation. (A) Alignment (using MultiAlign) of the 30 amino acid luminal domains of various KASH-domain NE proteins. The green bar denotes the highly conserved C-terminal prolines. (B) The tmNesprin-1, tmNesprin-2, tmNesprin-2P (lacks the last four aa) and dnNesprin-1 GFP fusion constructs used for the experiments shown in C-N. LD, luminal domain; SR, spectrin repeats; TM, transmembrane domain. (C-K) Dominant-negative effect of tmNesprin-1, tmNesprin-2 and dnNesprin-1 GFP fusions on the endogenous Nesprin proteins. Transiently transfected cells were fixed and subjected to immunofluorescence using the monoclonal K20-478 anti-Nesprin-2 and a rabbit polyclonal Nesprin-1 antibody. These antibodies did not recognise epitopes on the ectopically expressed polypeptides. Note the nuclear rim staining of endogenous Nesprin proteins in untransfected cells (arrowheads in E,H,K) and the absence of Nesprin staining in GFP-positive cells (arrows in E,H,K). (L-N) Confocal images demonstrate a cytoplasmic (panel L, arrows) and a diffuse nuclear staining pattern (panel L, arrowhead) for GFP-Nesprin-2P, which does not affect endogenous Nesprin-2 at the nuclear envelope (arrowhead in M). The cell lines used are indicated in the lower right-hand corner of the first column of frames (C,F,I). DNA was stained with DAPI. Images were obtained by confocal laser-scanning microscopy. Bars, 10 µm.

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