Molecular Structure and Assembly of Lamins in the context of Lamin-linked Disease

The lamins, nucleus-specific intermediate filament proteins, form a filamentous meshwork underneath the nuclear membrane and provide mechanical stability for eukaryotic nuclei. Lamina structures interact with numerous proteins in the nuclear membrane and the nucleoplasm, and with chromatin, and are thus essential for the dynamic functional organization of the nucleus. Relatively little is known about how lamins assemble and perform their functions on the molecular level. Mutations in lamins have recently been found to cause several heritable diseases termed laminopathies; the molecular mechanisms are unknown. The proposed studies focus on the analyses of lamin assembly in vitro and its relation to laminopathic diseases. As lamin assembly into bona fide 10nm filaments most likely requires interaction with lamin binding proteins of the nuclear membrane or on the chromatin surface, I will analyze the interaction of lamins with the membrane protein LAP2 beta, and with chromatin associated protein LAP2 alpha and narrow down the interaction domains. I will then focus on the analyses of lamin assembly and determine lamin domains important for assembly, and test the effect of LAP2 proteins on assembly. I also plan to establish a novel more physiological assembly assay on lipid or chromatin surfaces, mimicking the nuclear surrounding of lamins during assembly in vivo, in combination with atomic force and cryo-electron microscopy. Having established a suitable assay I will test the effect of various disease-linked lamin mutations on lamin interactions and assembly. Overall, the proposed work will develop a novel lamin assembly assay and test the influence of lamin-binding proteins and disease-specific mutations on lamin assembly and structure, thereby testing the "structural disease model" suggesting that lamin mutations disturb lamin structure and cause nuclear fragility.