Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins
Figure 4
F-actin interaction of Nesprin-1 ABD and Nesprin-3. (a) Nesprin-1 ABD binds to F-actin in vitro in the presence of Nesprin-3 in an F-actin co-sedimentation assay. Recombinantly expressed and purified GST-Nesprin-3 SR1,2,3 (GST NE3 SR1,2,3), and Nesprin-1 ABD (ABD NE1) were precleared and incubated with and without actin at room temperature under polymerizing conditions. High-speed centrifugation at 100,000 ×g for 1 h at 4°C was followed by separation of the protein mixture into supernatant (S) and pellet (P) fractions. ABD NE1 and GST NE3 SR1,2,3 bound to F-actin and were observed in the pellet fraction. F-actin binding was also observed when both proteins were added to the polymerization assay (actin + ABD NE1 + GST NE3 SR1,2,3). Samples were treated with 5x SDS-sample buffer and proteins separated on SDS-PA gels (12% acrylamide) and stained with Coomassie Brilliant Blue. The two closely migrating polypeptides in the Nesprin-1 ABD sample were identified as Nesprin-1 ABD by mass spectrometry. Arrows from top to bottom indicate the location of GST NE3 SR1,2,3, actin, and ABD NE 1. (b) Spectrin repeats of Nesprin-3 bind to F-actin. Nesprin-3 spectrin repeats SR1,2, SR2, and SR1 were used in the assay. Only SR1,2 (26.7 kDa) copelleted with F-actin (40 kDa) whereas SR2 (14.6 kDa) and SR1 (12.1 kDa) stayed in the supernatant. Proteins were separated on SDS-PA gels (18% acrylamide). The fast migrating protein in the SR1 sample corresponds also to SR1.
