Browsing School, Graduate by Subject "Lamins"
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A study of factors influencing the localization, sorting and assembly of the mammalian nuclear laminsIntermediate filament (IF) proteins are found in the cytoplasm as long 10 nm filaments and in the nucleus as a meshwork underlying the nuclear envelope. IF proteins contain a central conserved rod domain flanked by variable N- and C-terminal domains. At mitosis IFs disassemble, intermixing freely, but after mitosis the proteins resegregate to their respective compartments. In this work, three motifs, the nuclear localization signal (NLS), the CAAX motif, and the 42 AA insert, specific to lamin proteins, were studied as to their role in targeting and assembly. The NLS is believed to target lamins to the nucleus while the CAAX motif acts to confer association of lamins with the nuclear envelope. The 42 AA insert is thought to inhibit association of lamins with their cytoplasmic counterparts. To examine these theories HeLa cells were transiently transfected with IF molecules containing or lacking the three motifs and their cellular localization was studied. Chimeric molecules were also created from the cytoplasmic IF, NF-L, and lamin to further test the role of other IF domains in assembly and sorting. Lamin B mutants deleted of any one of the three motifs showed delayed but appropriate targeting to the nucleus. In contrast, mutants deleted of more than one motif were defective in targeting indicating that the three motifs act in concert with one another to ensure timely and correct lamina assembly. Stable cell lines expressing lamin B either with or without a CAAX motif were created. Biochemical analysis of the cell lines indicated that a large portion of lamin B does not co-fractionate with its putative receptor (p58), suggesting that the two proteins do not bind directly with one another. Overexpression of lamin mutants lacking a CAAX motif resulted in accumulation into multiple aggregates within the nucleus that did not colocalize with coiled bodies. Assembly phenotypes of IF mutants were dependent on the head domain sequences. Long filaments were formed with cytoplasmic head domains were present on lamin rods, but the converse phenotype of reticular filaments formed whenever the lamin head domain was present.