Summary ----------------------------------------------------------------------- The a-actinin superfamily is the largest of the F-actin cross-linking protein families Members of this superfamily have an actin-binding domain (ABD) consisting of a pair of calponin homology domains. We have cloned and characterized a novel giant actin-binding protein called Enaptin, which belongs to this family. Together with NUANCE, Enaptin forms a group of giant proteins that associate with the F-actin cytoskeleton as well as the nuclear membrane. Enaptin is composed of an N-terminal a-actinin type ABD, followed by a long coiled-coil rod and a transmembrane domain at the C-terminus. We have cloned and assembled a cDNA for Enaptin and found that the longest open reading frame of Enaptin encompasses 27,669 bp and predicts a 1014 kDa protein. The human Enaptin gene located on 6q23.1-25.3 spreads over 515 kb and gives rise to several splicing isoforms (Nesprin-1, Myne-1, Syne-1, CPG2). Northern blot analysis identified a >14 kb transcript and an additional transcript of 5.5 kb in brain. Using a polyclonal antibody against the ABD of Enaptin we detected a protein of approximately 400 kDa in tissues like brain and skeletal muscle. Further analysis showed that Enaptin is expressed in a wide range of tissues. Polyclonal antibodies generated against the C-terminus of Enaptin detected the protein at the nuclear envelope and in the cytosol of human fibroblasts. We showed that Enaptin is located in the outer nuclear membrane by selective permeabilisation of the plasma membrane with digitonin, furthermore its nuclear envelope localization was not affected by disruption of the actin cytoskeleton. Our studies also indicated that the nuclear envelope localization of Enaptin depended on lamin A/C. This is underlined by our findings in fibroblast cells from patients affected with laminopathies where we observed an altered distribution of Enaptin. With the N-terminal ABD domain and its C-terminal transmembrane domain, Enaptin has the potential to connect the nucleus to the actin cytoskeleton. Studies on Enaptin orthologues in lower eukaryotes proposed a role for these proteins in nuclear positioning and anchorage and in embryonic muscle development. Our myoblast differentiation studies showed an altered expression of Enaptin during differentiation. In order to reveal the functions of Enaptin and its link to human disease, we initiated a project for targeted disruption of Enaptin in the mouse