JCS029454 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Homology between the plectin-binding domain (PBD) of human nesprin-3 and mouse nesprin-3α and the KASH domains of human nesprin-1, -2, -3 and mouse nesprin-3α and -3β. (A) Basic structure of human (H-) nesprin-1, -2 and -3 and of mouse (M-) nesprin-3α and -3β showing the N-terminal PBD of human nesprin-3 and mouse nesprin-3α and the ABD of nesprin-1 and -2. The KASH domain is present at the C-terminus of all these proteins. (B) Alignment of mouse and human nesprin-3 and human nesprin-1 and -2 KASH sequences, showing a conserved homology (65%). The similar amino acids are shaded (gray).

• Supplemental Figure S2 -

  Fig. S2. Differential distribution of YFP-nesprin-3 and actin in control and DYT1 human fibroblasts. Primary fibroblasts from a control and DYT1 subject were infected with a lentivirus vector expressing YFP-nesprin-3 and fixed 72 hours later. The cells were immunostained for GFP and actin with merged images on the right. In infected control cells, YFP-nesprin-3 localized primarily to the perinuclear region and in both uninfected and infected cells, actin extended throughout the cytoplasm. In infected DYT1 cells, YFP-nesprin-3 localized in globular ER/NE structures (see Fig. 2), which were also enriched for β-actin; this was seen in two different cell lines of each type. Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Association of torsinB and YFP-nesprin-3. Human 293T cells were transfected with a plasmid encoding YFP-human-nesprin-3. Forty-eight hours later, cell lysates were immunoprecipitated with antibodies against torsinA and torsinB (TAB1) or specific to torsinB (TB2) in the presence of 1 mM ATP, as in Fig. 3. Proteins were resolved by SDS-PAGE and immunoblotted with goat antibodies to GFP or mouse antibodies to GAPDH.

• Supplemental Figure S4 -

  Fig. S4. TorsinA binds to both nesprin-3α and nesprin-3β. Human 293T cells were transfected with plasmid encoding expression cassettes for GFP-mouse-nesprin-3α or GFP-mouse-nesprin-3β. Forty-eight hours later, lysates were immunoprecipitated with antibodies to torsinA and proteins resolved by SDS-PAGE and immunoblotted with antibodies to GFP, as in Fig. 3.

• Supplemental Figure S5 -

  Fig. S5. Selective collapse of vimentin cytoskeletal network by PMA treatment. Control human fibroblasts were left untreated or exposed to PMA for 30 minutes and the distribution of vimentin and actin (phalloidin) (A-F), and of vimentin and tubulin (G-L), was evaluated by immunocytochemistry (as in Fig. 7). Scale bar: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Nuclear integrity in torsinA^−/− MEFs. Electron micrograph showing the NE in a typical torsinA^−/− MEF. Scale bar: 500 nm.