Molecular analysis of nuclear lamin assembly

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Project Summary Lamin filaments are central structural organizers of the metazoan nucleus. They contribute to nuclear function by controlling nuclear structure, separating the nucleoplasm from the cytoplasm and organizing the genome into differentially regulated subdomains. Many diseases are associated with lamin dysregulation and abnormal nuclear structure, underscoring the importance of these molecules. Despite the importance of lamins in normal nuclear function, molecular mechanisms controlling lamin assembly are poorly understood. Previous studies attempted to dissect lamin assembly using recombinant lamin proteins purified under denaturing conditions and simultaneously refolded and assembled into filamentous structures through removal of denaturant. It is now clear that the lamin structures assembled in these experiments do not resemble lamin filaments in cells. The goal of this proposal is to develop experimental systems for studying physiological lamin assembly and to determine the assembly pathway and mechanism of lamin assembly. The research is expected to extend understanding of nuclear structure and function, and of diseases associated with dysregulation of nuclear structure and function. The proposed experiments aim to uncover molecular mechanisms of lamin assembly. In vitro experiments will be conducted in Xenopus laevis egg extracts, which contain their own soluble lamin protein, eliminating the need for recombinant lamins in assembly assays. Xenopus egg extracts can assemble diverse lamin structures. By varying assembly conditions and studying these lamin assemblies using fluorescence and electron microscopy, cellular structures and signals that control lamin assembly will be identified. Using analytical biochemistry, the soluble lamin subunit will be characterized, along with any proteins that are in a stable complex with the soluble lamin subunit. Importin  and  are known binding partners of soluble lamin in Xenopus egg extract. Proposed experiments will determine how importins and other lamin-binding proteins regulate lamin assembly. In vivo experiments will be conducted in genome edited stem cells. Mouse embryonic stem cells with the genes encoding all three lamin isoforms knocked out have been isolated and propagated by the host lab. Inducibly expressing fluorescently tagged lamin in these cells is predicted to result in nascent lamin meshwork assembly, allowing visualization of the succession of lamin assembly using fluorescence and electron microscopy. By comparing assembly of fluorescently tagged lamin mutants to assembly of wild type lamins, the research will determine whether the lamin assembly pathway is altered by disease-causing lamin mutations. The research proposed will be conducted in the laboratory of Dr. Yixian Zheng at the Carnegie Institution for Science Department of Embryology. Research will be carried out independently with biweekly guidance provided by Dr. Zheng. Experimental training, along with training in science writing and presentation, will be accomplished through one-on-one interactions between Dr. Zheng and the trainee and through participation in the collegial, collaborative, and interactive environment of the Carnegie Institution.