The nuclear lamins form a lattice of intermediate filaments on the nucleoplasmic surface of the nuclear envelope and reversibly assemble and disassemble during the cell cycle. This is a proposal to determine the domains of the nuclear lamin proteins that function in nuclear transport, assembly into the nuclear envelope, processing, and disassembly in metaphase. In addition, experiments will be performed to uncover the role of the nuclear lamins in nuclear pore distribution and interphase chromosome organization. These questions will be approached by making both random and site-specific mutations in the coding regions of the human lamin cDNAs and expressing them in CHO cells. The human lamins will be detected and distinguished from the endogenous CHO lamins by using a human-specific monoclonal antibody. Preliminary experiments show that the human A and C lamin proteins can be expressed in CHO cells and that they are correctly transported into the nucleus, assembled into the nuclear envelope, processed, and disassembled in metaphase. We have produced both dominant and recessive effects on these processes by making in-frame insertional mutations in the A-lamin cDNA. The independent effects of these mutations suggest that they may be the result of local perturbations of the lamin proteins, and that more discrete experiments might define these regions as functional domains. The ability to assay assembly and disassembly will provide a means of testing the coiled-coil theory, understanding the function of the pre-A lamin processing, and how phosphorylation disassembles the nuclear envelope. Additional mutant phenotypes might provide clues about the actual functions of the nuclear envelope and about cellular regulation in general.
| Heald, R; McLoughlin, M; McKeon, F (1993) Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell 74:463-74 |
| Loewinger, L; McKeon, F (1988) Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm. EMBO J 7:2301-9 |
